DNA molecules between telepathy
The latest study, DNA molecules appear to have telepathy. Scientists have discovered the double helix structure of DNA molecules to identify themselves with the "matching" elements, even some distance away, on the surface and no other outside help, the match of the two elements together to the end. In accordance with the previous understanding of DNA, scientists study the double helix structure of DNA molecules are arranged in accordance with the laws of their own. Helix structure of DNA molecule is composed of many ODN from the polymerization of long-chain, as the composition of the DNA base pairs of only four: adenine (A), guanine (G), cytosine (C) And thymine (T), therefore, there are four types of DNA, we usually A, T, C, G Four alphabet tag them, and use of chemical methods to match their children - A equipped with T, C equipped with G . In fact, the well-known "to exchange the role of the base together" is not the DNA double helix molecule in close connection with the bodies of the root causes. The DNA double helix structure was so stable, because the outside of deoxyribose phosphate and arranged in alternating the basic framework, the inside of the base to form hydrogen bonds through the base right. Scientists study the mixture through the fluorescence of this double-stranded DNA molecule structure. These DNA molecules were placed on some of the salt water, salt water test does not contain any proteins, DNA molecules will enable non-binding, as well as any material that may affect the trial. Strangely enough, together with the same number of base pairs of DNA molecules is the other remaining twice the number of DNA molecules. Even though they look like a very strange, like a psychological sense, but in fact only a DNA molecule in the exercise under the laws of physics, not a supernatural phenomenon. Take charge of deoxyribose and phosphate arranged in turn composed of DNA molecules will be mutually exclusive, however, because of the DNA double helix structure of the special, making the repulsive force between them to reach the minimum. In order to understand more vividly the researchers said, let us try the double helix structure of DNA molecules into a corkscrew imagination to form a long chain of DNA molecules in the base of support outside the framework and the role of hydrogen bonds in the middle of, So that the screw cone to the direction of a distorted, twisted into a spiral, then the process will be part of the same degree of bending and other elements of the sunken part of the coordinated combination. Scientists point out that this "psychological sense" would help the DNA molecules in the chaos of their pre-arranged neatly, which can effectively avoid errors occur when the combination of DNA, it effectively avoiding cancer, aging and other diseases. However, due to the same DNA sequence in fact disrupt the combination of sexual reproduction is a meaningful, because of the need to ensure that future generations have the genetic diversity.
Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location
Environmental and Occupational Toxicology Division, HECSB, Ottawa, ON, Canada K1A 0K9; Department of Biology, McMaster University, 1280 Main Street West, Hamilton, ON, Canada L8S 4K1; ¶Nutrition and Toxicology Research Institute Maastricht, NUTRIM, Department of Health Risk Analysis and Toxicology, Maastricht University, 6200 MD, PO Box 616, Maastricht, The Netherlands; Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, AR 72079; **Department of Biological Sciences, University of Lethbridge, 4401 University Drive, Lethbridge, Alta., Canada T1K 3M4; and Biostatistics and Epidemiology Division, Healthy Environments and Consumer Safety Branch, Ottawa, ON, Canada K1A 0K9
Edited by James E. Cleaver, University of California, San Francisco, CA, and approved November 20, 2007 (received for review June 25, 2007)
Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.
Edited by James E. Cleaver, University of California, San Francisco, CA, and approved November 20, 2007 (received for review June 25, 2007)
Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.
Direct Visualization of the EcoRII-DNA Triple Synaptic Complex by Atomic Force Microscopy
Interactions between distantly separated DNA regions mediated by specialized proteins lead to the formation of synaptic protein-DNA complexes. This is a ubiquitous phenomenon which is critical in various genetic processes. Although such interactions typically occur between two sites, interactions among three specific DNA regions have been identified, and a corresponding model has been proposed. Atomic force microscopy was used to test this model for the EcoRII restriction enzyme and provide direct visualization and characterization of synaptic protein-DNA complexes involving three DNA binding sites. The complex appeared in the images as a two-loop structure, and the length measurements proved the site specificity of the protein in the complex. The protein volume measurements showed that an EcoRII dimer is the core of the three-site synaptosome. Other complexes were identified and analyzed. The protein volume data showed that the dimeric form of the protein is responsible for the formation of other types of synaptic complexes as well. The applications of these results to the mechanisms of the protein-DNA interactions are discussed
DNA sequencing gels
Gels used for DNA sequence analysis are of the wedge type. These produce a voltage gradient which decreases as DNA migrates down the gel, thus retarding the rate of migration of smaller fragments and allowing more readable sequence information to be obtai ned from one gel. DNA sequencing gels are cast between the 38 x 50cm and 38 x 47.5cm glass plates of the Bio-Rad SequiGen?sequencing system.
You will need:
A standard detergent2% dichlorodimethyl silane in hexaneAbsolute ethanol6% sequencing acrylamide (5.7% acrylamide, 0.3% bisacrylamide, 48% urea, 1x TBE)25% AMPS (freshly made)TEMED
N.B: Wear gloves while handling solutions of unpolymerised acrylamide. Unpolymerised acrylamide is a neurotoxin.
1) Clean the glass plates extensively with detergent and water, tap water, distilled water and finally ethanol. Wipe dry with a clean paper towel.
2) Siliconize the smaller of the two plates using the 4% solution of dichlorodimethylsilane in hexane. The solution should be spread evenly over the plate and allowed to dry before being repeated. Once dry, the plate should be washed with 100% ethanol and again wiped dry using a clean paper towel.
3) Gel plates are then assembled as described in the manufacturers instructions using two 0.25 -1mm wedge spacers.
Polyacrylamide sequencing mix for use in the gels was stored at 4°C in a dark bottle.
4) 35ml of the acrylamide mix is used to first plug the bottom of the gel. Chill the acrylamide on ice and add 150ul 25% AMPS and 150ul TEMED. Mix by swirling and then poured briskly into the gel mould. The quantities of AMPS and TEMED may have to be esti mated empirically to cause setting in approx. 5 minutes.
5) Once the plug has set, 85ml of acrylamide is then used to form the main gel itself. To the acrylamide (chilled on ice beforehand), add 110ul 25% AMPS and 110ul TEMED. The solutions are mixed thoroughly, placed into a 50ml syringe and injected, carefull y, between the glass plates. In order to facilitate ease of pouring, the glass plates were inclined at an angle of approximately 10° to the horizontal in a large developing tray to prevent spills. Again, the quantities of AMPS and TEMED used may need to be varied in order to give polymerisation in approx. 30 minutes - this may be especially critical if the ambient temperature is abnormally warm.
N.B: It is critical to chill the acrylamide for the main gel in order to prevent polymerisation while the gel is being poured. You may also need to adjust the AMPS/TEMED quantities used. You should aim to have the plug set in ~5 mins and the main g el after ~30 mins.
6) Immediately after the gel is poured, a flat 0.25mm spacer (or reversed shark tooth comb) should be placed into the acrylamide on the gel top such that it intrudes into the gel by approximately 10mm. This allows the formation of a flat gel surface essen tial to the effective use of the shark tooth combs during electrophoresis. Clamp large bulldog clips across the top of the gel plates during gel polymerisation to ensure a leak-free fit of the combs. Allowed to polymerise for 1 hour at room temperature an d then use directly or store overnight at 4°C, tightly wrapped in clingfilm to prevent dehydration of the gel.
7) Remove the gel former and pre-electrophorese the gel at 1800V to heat the gel and running buffer to the required operating temperature (55°C) prior to the loading of the samples. Running buffer is 1 x TBE.
8) Insert sharks tooth combs such that the tips protrude approximately 0.5mm into the gel surface.
9) Thoroughly wash the wells immediately prior to the loading of the samples with running buffer to remove any urea which leaches from the gel.
10) Sequencing reaction mixtures, containing loading buffer, should be boiled for 2-3 minutes to denature any secondary structure and loaded into the wells (3ul/well), in the order G, A, T, C.
11) Electrophorese at 1800V (preferably 75W constant power) until the xylene cyanol dye front is approximately 5 cm from the bottom of the gel. Monitor the gel temperature to ensure it stays at 60°C or below (preferably 50-55°C).
N.B: Allowing the gel temperature to exceed 60°C for extended periods of time will cause the hydrolysis of urea in the gel.
12) After electrophoresis is complete, combs should be removed and the small siliconized glass plate gently removed from the remaining plate. The large plate, with the gel still attached, is then immersed in a fixative solution containing 10% acetic acid, 10% methanol for approximately 15-20 minutes. This process is used to remove urea from the gel.
13) Transfer the gel to a large sheet of Whatman 3MM paper and dry on a vacuum gel drier at 85°C for 75 minutes prior to autoradiography.
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Addendum submitted at 17:32 on 08/02/96 by: Dr. Simon Dawson,Department of Biochemistry,University of Nottingham,The Medical School,Q.M.C.,Clifton Boulevard,Nottingham,NG7 2UH,U.K.Tel: +44 115 9249924 Ex. 44787,FAX: +44 115 9422225,Email: Simon.Dawson@nott.ac.uk
This addendum describes the use of formamide in sequencing gels to alleviate problems with base stacking due to G-C compressions. The following method describes the production of 200ml of a 25% formamide, 6% acrylamide sequencing gel mix but it can be scaled accordingly. You can also vary the formamide concentration upto 40%.
You will need:
Ultra-pure ureaFormamideAcrylamideBis-acrylamideTEMEDAmmonium persulphate (AMPS)Amberlite MB-1 resin (Sigma)10 x TBE buffer
1) Mix together 82g urea, 50ml formamide, 11.4g acrylamide, 0.6g bis-acrylamide and ~60ml H2O.
2) Warm the mixture to ~40°C, with stirring, to dissolve solids.
N.B.: Do not heat the mixture above 55°C as this leads to hydrolysis of the urea.
3) Once dissolved, add ~5g Amberlite MB-1 resin and stir for 20 minutes.
4) Filter solution (we typically use Whatman 3MM paper) and make up to 180ml with H2O.
5) Add 20ml fresh 10 x TBE buffer.
6) For a gel of ~100ml, use 200ul TEMED and 200ul fresh 25% AMPS solution to initiate polymerisation.
N.B.: For the above quantities of TEMED and AMPS, you MUST chill the gel mix to ~4°C prior to addition of the catalysts - this prevents gel polymerisation mid-way through pouring the gel! Polymerisation of formamide gels requires longer than normal as does the actual electrophoresis.
7) Electrophorese at ~80W, constant power (should be ~45 - 50°C for a 38x50cm gel).
You will need:
A standard detergent2% dichlorodimethyl silane in hexaneAbsolute ethanol6% sequencing acrylamide (5.7% acrylamide, 0.3% bisacrylamide, 48% urea, 1x TBE)25% AMPS (freshly made)TEMED
N.B: Wear gloves while handling solutions of unpolymerised acrylamide. Unpolymerised acrylamide is a neurotoxin.
1) Clean the glass plates extensively with detergent and water, tap water, distilled water and finally ethanol. Wipe dry with a clean paper towel.
2) Siliconize the smaller of the two plates using the 4% solution of dichlorodimethylsilane in hexane. The solution should be spread evenly over the plate and allowed to dry before being repeated. Once dry, the plate should be washed with 100% ethanol and again wiped dry using a clean paper towel.
3) Gel plates are then assembled as described in the manufacturers instructions using two 0.25 -1mm wedge spacers.
Polyacrylamide sequencing mix for use in the gels was stored at 4°C in a dark bottle.
4) 35ml of the acrylamide mix is used to first plug the bottom of the gel. Chill the acrylamide on ice and add 150ul 25% AMPS and 150ul TEMED. Mix by swirling and then poured briskly into the gel mould. The quantities of AMPS and TEMED may have to be esti mated empirically to cause setting in approx. 5 minutes.
5) Once the plug has set, 85ml of acrylamide is then used to form the main gel itself. To the acrylamide (chilled on ice beforehand), add 110ul 25% AMPS and 110ul TEMED. The solutions are mixed thoroughly, placed into a 50ml syringe and injected, carefull y, between the glass plates. In order to facilitate ease of pouring, the glass plates were inclined at an angle of approximately 10° to the horizontal in a large developing tray to prevent spills. Again, the quantities of AMPS and TEMED used may need to be varied in order to give polymerisation in approx. 30 minutes - this may be especially critical if the ambient temperature is abnormally warm.
N.B: It is critical to chill the acrylamide for the main gel in order to prevent polymerisation while the gel is being poured. You may also need to adjust the AMPS/TEMED quantities used. You should aim to have the plug set in ~5 mins and the main g el after ~30 mins.
6) Immediately after the gel is poured, a flat 0.25mm spacer (or reversed shark tooth comb) should be placed into the acrylamide on the gel top such that it intrudes into the gel by approximately 10mm. This allows the formation of a flat gel surface essen tial to the effective use of the shark tooth combs during electrophoresis. Clamp large bulldog clips across the top of the gel plates during gel polymerisation to ensure a leak-free fit of the combs. Allowed to polymerise for 1 hour at room temperature an d then use directly or store overnight at 4°C, tightly wrapped in clingfilm to prevent dehydration of the gel.
7) Remove the gel former and pre-electrophorese the gel at 1800V to heat the gel and running buffer to the required operating temperature (55°C) prior to the loading of the samples. Running buffer is 1 x TBE.
8) Insert sharks tooth combs such that the tips protrude approximately 0.5mm into the gel surface.
9) Thoroughly wash the wells immediately prior to the loading of the samples with running buffer to remove any urea which leaches from the gel.
10) Sequencing reaction mixtures, containing loading buffer, should be boiled for 2-3 minutes to denature any secondary structure and loaded into the wells (3ul/well), in the order G, A, T, C.
11) Electrophorese at 1800V (preferably 75W constant power) until the xylene cyanol dye front is approximately 5 cm from the bottom of the gel. Monitor the gel temperature to ensure it stays at 60°C or below (preferably 50-55°C).
N.B: Allowing the gel temperature to exceed 60°C for extended periods of time will cause the hydrolysis of urea in the gel.
12) After electrophoresis is complete, combs should be removed and the small siliconized glass plate gently removed from the remaining plate. The large plate, with the gel still attached, is then immersed in a fixative solution containing 10% acetic acid, 10% methanol for approximately 15-20 minutes. This process is used to remove urea from the gel.
13) Transfer the gel to a large sheet of Whatman 3MM paper and dry on a vacuum gel drier at 85°C for 75 minutes prior to autoradiography.
--------------------------------------------------------------------------------
Addendum submitted at 17:32 on 08/02/96 by: Dr. Simon Dawson,Department of Biochemistry,University of Nottingham,The Medical School,Q.M.C.,Clifton Boulevard,Nottingham,NG7 2UH,U.K.Tel: +44 115 9249924 Ex. 44787,FAX: +44 115 9422225,Email: Simon.Dawson@nott.ac.uk
This addendum describes the use of formamide in sequencing gels to alleviate problems with base stacking due to G-C compressions. The following method describes the production of 200ml of a 25% formamide, 6% acrylamide sequencing gel mix but it can be scaled accordingly. You can also vary the formamide concentration upto 40%.
You will need:
Ultra-pure ureaFormamideAcrylamideBis-acrylamideTEMEDAmmonium persulphate (AMPS)Amberlite MB-1 resin (Sigma)10 x TBE buffer
1) Mix together 82g urea, 50ml formamide, 11.4g acrylamide, 0.6g bis-acrylamide and ~60ml H2O.
2) Warm the mixture to ~40°C, with stirring, to dissolve solids.
N.B.: Do not heat the mixture above 55°C as this leads to hydrolysis of the urea.
3) Once dissolved, add ~5g Amberlite MB-1 resin and stir for 20 minutes.
4) Filter solution (we typically use Whatman 3MM paper) and make up to 180ml with H2O.
5) Add 20ml fresh 10 x TBE buffer.
6) For a gel of ~100ml, use 200ul TEMED and 200ul fresh 25% AMPS solution to initiate polymerisation.
N.B.: For the above quantities of TEMED and AMPS, you MUST chill the gel mix to ~4°C prior to addition of the catalysts - this prevents gel polymerisation mid-way through pouring the gel! Polymerisation of formamide gels requires longer than normal as does the actual electrophoresis.
7) Electrophorese at ~80W, constant power (should be ~45 - 50°C for a 38x50cm gel).
DNA Sequencing
The sequencing reactions described below work perfectly well if you are short of cash to buy sequencing kits. It is based on the Dideoxy sequencing method of Sanger et al., 1977. However, due to the number solutions that need to be made, I recommend purch asing a sequencing kit, we use either the T7 Sequencing Kit (Pharmacia, 100 reactions) or the Sequenase 2.0 Sequencing Kit (USB via Amersham in the U.K., 100 reactions). Reactions are performed in sterile 1.5ml microcentrifuge tubes. Primers are synthesised on an Applied Biosystems 381A DNA synthesiser.
Approximately 3ug denatured, high quality dsDNA (i.e. prepared as described in 'Plasmid Isolation using PEG') are typically used for standard sequencing reactions.
DNA Sequencing Reactions
You will need:
Freshly made 2M NaOH3M sodium acetate, pH 4.5Sterile, distilled waterAbsolute ethanol70% ethanol7 x DNA annealing buffer (280mM Tris.Cl, pH 7.5, 100mM MgCl2, 350mM NaCl)Termination mixes (40mM Tris.Cl, pH 7.5, 50mM NaCl, 10mM MgCl2, 150mM dTTP, 150mM dATP, 150mM dCTP, 150mM c7deaza-dGTP and 15mM of the respective ddNTP)5 x DNA labelling mix (10mM dGTP, 10mM dCTP, 10mM dTTP, 200mM Tris.Cl, pH 7.5, 250mM NaCl)300mM DTT[a-35S]-dATP (~ 1000Ci/mmol, Amersham or DuPont)T7 DNA polymerase (Pharmacia)Stop solution (95% deionized formamide, 20mM EDTA, pH 7.5, 0.1% each of bromophenol blue and xylene cyanol FF)
1) Denature dsDNA by the addition of 8ul DNA (approximately 3ug) to a sterile microcentrifuge tube containing 2ul freshly made 2M NaOH vortexed briefly and incubate at room temperature for 10 minutes.
2) Neutralise DNA by the addition of 3ul 3M sodium acetate, pH 4.5 and 7ul sterile, distilled H2O and precipitate by the addition of 60ul ethanol. Recover DNA by centrifugation, at maximum speed, for 10 minutes in a microfuge. Rinse DNA briefly in 70% ethanol, air dry and re-dissolve in 10ul sterile, distilled H2O.
3) To a microcentrifuge tube containing 10ml denatured template DNA, add 4.44ng primer (2ul of a 2.22ng/ul stock) and 2ml 7 x annealing buffer. Heat the mixture to 65°C for 2 minutes and allow to cool slowly, over a period of about 30 minutes, to roo m temperature.
4) While the annealing reaction is taking place, take 4 sterile microcentrifuge tubes per sample and label G, A, T, C, respectively. Place into each tube 2.5ul, respectively, of the corresponding termination mix. Pre-warm tubes to 37°C.
5) After completion of the annealing reaction, the labelling reaction is initiated by the addition to the annealed template/primer of 2ul 1 x labelling mix, 1ul 300mM DTT, 1ul [a-35S]-dATP (~ 1000Ci/mmol) and 3 units T7 DNA polymerase (2ul of a 1.5 units/ ul solution). The solution is pipetted briefly to mix the components and incubated at 4°C for 2-5 minutes.
6) Termination is achieved by transferring 4.5ul of the labelling reaction into each of the 4 tubes labelled G, A, T, C, respectively and incubating at 37°C for 2-5 minutes.
7) After termination, 5ul stop solution should be added to each tube, mixed by pipetting and the samples stored at -20°C for later use.
Approximately 3ug denatured, high quality dsDNA (i.e. prepared as described in 'Plasmid Isolation using PEG') are typically used for standard sequencing reactions.
DNA Sequencing Reactions
You will need:
Freshly made 2M NaOH3M sodium acetate, pH 4.5Sterile, distilled waterAbsolute ethanol70% ethanol7 x DNA annealing buffer (280mM Tris.Cl, pH 7.5, 100mM MgCl2, 350mM NaCl)Termination mixes (40mM Tris.Cl, pH 7.5, 50mM NaCl, 10mM MgCl2, 150mM dTTP, 150mM dATP, 150mM dCTP, 150mM c7deaza-dGTP and 15mM of the respective ddNTP)5 x DNA labelling mix (10mM dGTP, 10mM dCTP, 10mM dTTP, 200mM Tris.Cl, pH 7.5, 250mM NaCl)300mM DTT[a-35S]-dATP (~ 1000Ci/mmol, Amersham or DuPont)T7 DNA polymerase (Pharmacia)Stop solution (95% deionized formamide, 20mM EDTA, pH 7.5, 0.1% each of bromophenol blue and xylene cyanol FF)
1) Denature dsDNA by the addition of 8ul DNA (approximately 3ug) to a sterile microcentrifuge tube containing 2ul freshly made 2M NaOH vortexed briefly and incubate at room temperature for 10 minutes.
2) Neutralise DNA by the addition of 3ul 3M sodium acetate, pH 4.5 and 7ul sterile, distilled H2O and precipitate by the addition of 60ul ethanol. Recover DNA by centrifugation, at maximum speed, for 10 minutes in a microfuge. Rinse DNA briefly in 70% ethanol, air dry and re-dissolve in 10ul sterile, distilled H2O.
3) To a microcentrifuge tube containing 10ml denatured template DNA, add 4.44ng primer (2ul of a 2.22ng/ul stock) and 2ml 7 x annealing buffer. Heat the mixture to 65°C for 2 minutes and allow to cool slowly, over a period of about 30 minutes, to roo m temperature.
4) While the annealing reaction is taking place, take 4 sterile microcentrifuge tubes per sample and label G, A, T, C, respectively. Place into each tube 2.5ul, respectively, of the corresponding termination mix. Pre-warm tubes to 37°C.
5) After completion of the annealing reaction, the labelling reaction is initiated by the addition to the annealed template/primer of 2ul 1 x labelling mix, 1ul 300mM DTT, 1ul [a-35S]-dATP (~ 1000Ci/mmol) and 3 units T7 DNA polymerase (2ul of a 1.5 units/ ul solution). The solution is pipetted briefly to mix the components and incubated at 4°C for 2-5 minutes.
6) Termination is achieved by transferring 4.5ul of the labelling reaction into each of the 4 tubes labelled G, A, T, C, respectively and incubating at 37°C for 2-5 minutes.
7) After termination, 5ul stop solution should be added to each tube, mixed by pipetting and the samples stored at -20°C for later use.
DNA Fragmentation Assay via Dipheylamine
Protocol I: Triton X-100 Lysis Buffer
In 96 flat-wells plate, incubate 4x10 6 target cells (40 wells of 105 per well) with desired concentration of effectors (105 target cells per well). After incubation, collect the cell sample in 1.5 ml eppendorf tube, spin down, resuspend with 0.5 ml PBS in 1.5 ml eppendorf tubes, and add 55ul of lysis buffer for 20 min on ice (4oC). Centrifuge the eppendorf tubes in cold at 12,000 g for 30 minutes. Transfer the samples to new 1.5 ml eppendorf tubes and then extract the supernatant with 1:1 mixture of phenol:chloroform (gentle agitation for 5 min followed by centrifugation) and precipitate in two equivalence of cold ethanol and one-tenth equivalence of sodium acetate. Spin down, decant, and resuspend the precipitates in 30ul of deionized water-RNase solution (0.4ml water + 5ul of RNase) and 5ul of loading buffer for 30 minutes at 37oC. Also insert 2ul of Hindi III marker (12ul of Stock IV) on the outer lanes. Run the 1.2% gel at 5V for 5min before increasing to 100V.
Protocol II: SDS LysisBuffer
Add SDS lysis buffer to the incubated cell samples (prepared as in Protocol I).
Stock I:Triton X-100 Lysis Buffer 40 ml of 0.5 M EDTA 5 ml of 1 M TrisCl buffer pH 8.0 5 ml of 100% Triton X-100 50 ml of H2OStock II: SDS Lysis Buffer
Stock III: 1.2% Agarose Gel
Prepare a stock of 2 liter of 1X TAE (i.e., 2 liter + 40ml of 5X TAE). Add 2.4g of agarose power(1.2% agarose) to 200ml of 1X TAE solution and microwave for 4 min at high power. Then cool the gel to 50oC and add 25ul of ethium bromide before pouring it into the gel plate. Insert comb and let the gel polymerized.
Stock IV: Hindi III Marker (50 Kb lamda DNA) 4ul of Hindi III Marker 16ul of Deionized Water 4ul of Loading Buffer
Protocol III: DNA Fragmentation Assay via Dipheylamine
In 24-wells plate, incubate 5 X 106 targets with desired number of effectors. After incubation, transfer the samples to 15ml tubes, centrifuge for 30 s at 1500g, and resuspend in 5ml of lysis buffer (Stock IV) for 15 min on ice. Centrifuge the samples for 20 min at 27,000g to separate high-molecular-weight chromatin from cleavage products. Resuspend the pellet in 5 ml of buffer (stock V).
reat the supernatants and pellets with the diphenylamine reagent (Stock VI) and incubate at 370C for 16-24 hr before colorimetric assessment.
Stock IV: Lysis buffer at pH 8.0 5mM Tris-HCl 20mM EDTA 0.5% Triton X-100
Stock V: Buffer at pH 8.0 10mM Tris-HCl 1mM EDTA
Stock VI: Diphenylamine reagent (light sensitive) 1.5g of diphenylamine (steam-distilled) 100ml acetic acid (redistilled) 1.5ml of conc. sulfuric acid
On the day of usage, add 0.10ml of ag acetaldehyde (16mg/ml) to 20ml of the diphenylamine reagent.
Protocol IV: DNA Fragmentation via 3H-TdR
5 X 106 target cells were labeled with 50µl of 3H-TdR (1 mCi/ml) overnight in 10 ml of media. The next day, the cells were washed 3X with 10ml of PBS and incubated in 10ml of media to chase out unincorporated cytoplasmic 3H-TdR. After incubating for 2 hrs, the cells were washed 3X with PBS and then used in lytic assay under the same conditions as the 51Cr release assay in 96 v-well plates. At the end of the assay, each well was treated with 20µl of 1.0% Triton-X on ice for 5 minutes, followed by centrifugation at 1500g in a Beckman T-J6 rotor for 15 minutes. 100µl of the supernatant were harvested from each well and counted in a scintillation counter. Total count was obtained by resuspending the cells prior to harvesting, and adding 0.1% SDS to solublilize the cells. The % 3H released was calculated with an equation analogous to that for %51Cr released.
In 96 flat-wells plate, incubate 4x10 6 target cells (40 wells of 105 per well) with desired concentration of effectors (105 target cells per well). After incubation, collect the cell sample in 1.5 ml eppendorf tube, spin down, resuspend with 0.5 ml PBS in 1.5 ml eppendorf tubes, and add 55ul of lysis buffer for 20 min on ice (4oC). Centrifuge the eppendorf tubes in cold at 12,000 g for 30 minutes. Transfer the samples to new 1.5 ml eppendorf tubes and then extract the supernatant with 1:1 mixture of phenol:chloroform (gentle agitation for 5 min followed by centrifugation) and precipitate in two equivalence of cold ethanol and one-tenth equivalence of sodium acetate. Spin down, decant, and resuspend the precipitates in 30ul of deionized water-RNase solution (0.4ml water + 5ul of RNase) and 5ul of loading buffer for 30 minutes at 37oC. Also insert 2ul of Hindi III marker (12ul of Stock IV) on the outer lanes. Run the 1.2% gel at 5V for 5min before increasing to 100V.
Protocol II: SDS LysisBuffer
Add SDS lysis buffer to the incubated cell samples (prepared as in Protocol I).
Stock I:Triton X-100 Lysis Buffer 40 ml of 0.5 M EDTA 5 ml of 1 M TrisCl buffer pH 8.0 5 ml of 100% Triton X-100 50 ml of H2OStock II: SDS Lysis Buffer
Stock III: 1.2% Agarose Gel
Prepare a stock of 2 liter of 1X TAE (i.e., 2 liter + 40ml of 5X TAE). Add 2.4g of agarose power(1.2% agarose) to 200ml of 1X TAE solution and microwave for 4 min at high power. Then cool the gel to 50oC and add 25ul of ethium bromide before pouring it into the gel plate. Insert comb and let the gel polymerized.
Stock IV: Hindi III Marker (50 Kb lamda DNA) 4ul of Hindi III Marker 16ul of Deionized Water 4ul of Loading Buffer
Protocol III: DNA Fragmentation Assay via Dipheylamine
In 24-wells plate, incubate 5 X 106 targets with desired number of effectors. After incubation, transfer the samples to 15ml tubes, centrifuge for 30 s at 1500g, and resuspend in 5ml of lysis buffer (Stock IV) for 15 min on ice. Centrifuge the samples for 20 min at 27,000g to separate high-molecular-weight chromatin from cleavage products. Resuspend the pellet in 5 ml of buffer (stock V).
reat the supernatants and pellets with the diphenylamine reagent (Stock VI) and incubate at 370C for 16-24 hr before colorimetric assessment.
Stock IV: Lysis buffer at pH 8.0 5mM Tris-HCl 20mM EDTA 0.5% Triton X-100
Stock V: Buffer at pH 8.0 10mM Tris-HCl 1mM EDTA
Stock VI: Diphenylamine reagent (light sensitive) 1.5g of diphenylamine (steam-distilled) 100ml acetic acid (redistilled) 1.5ml of conc. sulfuric acid
On the day of usage, add 0.10ml of ag acetaldehyde (16mg/ml) to 20ml of the diphenylamine reagent.
Protocol IV: DNA Fragmentation via 3H-TdR
5 X 106 target cells were labeled with 50µl of 3H-TdR (1 mCi/ml) overnight in 10 ml of media. The next day, the cells were washed 3X with 10ml of PBS and incubated in 10ml of media to chase out unincorporated cytoplasmic 3H-TdR. After incubating for 2 hrs, the cells were washed 3X with PBS and then used in lytic assay under the same conditions as the 51Cr release assay in 96 v-well plates. At the end of the assay, each well was treated with 20µl of 1.0% Triton-X on ice for 5 minutes, followed by centrifugation at 1500g in a Beckman T-J6 rotor for 15 minutes. 100µl of the supernatant were harvested from each well and counted in a scintillation counter. Total count was obtained by resuspending the cells prior to harvesting, and adding 0.1% SDS to solublilize the cells. The % 3H released was calculated with an equation analogous to that for %51Cr released.
DNA Fingerprinting
Perhaps one of the most disquieting elements in our justice system today is the thought of wrongfully convicting or even sentencing to death the innocent. Biochemistry, in the development of DNA forensics, provides justice a more decisive scale in weighing the innocence or guilt of an individual.
In 1985 Ronald Cotton was imprisoned for the rape of Jennifer Thompson. She had identified him from pictures and a line-up as the assailant. Circumstantial evidence stacked up against Cotton making it quite clear that Cotton was guilty. He was imprisoned with no flaw in the judicial system to speak of, except one…he was innocent. After 10 years of imprisonment, biochemical technology allowed the comparison of Cotton’s DNA to that of the rapist’s semen, proving his innocence beyond a shadow of a doubt. The same evidence was then used to rightfully convict Robert Poole, a convict who had mentioned the rape to fellow inmates.Since the early 1980’s DNA "fingerprints" have been used to convict or release possible suspects involved in many crimes. The use of these fingerprints by the FBI’s Forensic Science Systems Unit to form the national DNA registry called, Combined DNA Index System (CODIS), is perhaps one of the greatest assets to criminal investigators to date. It is the goal of the FBI to have the Index operating much like the Automated Fingerprint Index System (AFIS). The Standardization Project has insured that all of the genetic information collected across the United States is in a comparable format. All states have authorized the submission of DNA from violent criminals and sexual offenders to CODIS. Almost any type of biological evidence found at a crime scene may be compared to a suspect, including: blood, semen, saliva, bone, tissue, teeth, and even hair follicles.
he methods for obtaining and comparing the genetic samples of evidence and suspects were originally developed to determine the compatibility of Human Leukocyte Antigens (HLA) in individuals for organ and bone marrow transplants. These antigens are highly specific to an individual and are used by the body’s immune system to determine self from non-self. If a transplant is made without first comparing the compatibility of these antigens, there is a very high likelihood that the donated tissue will be rejected and attacked by the recipient’s immune system. The high amount of specificity allows the regions of DNA encoding for the antigens to be extremely useful in identifying one out of several million people. The commonly used methods for isolating and comparing genetic sequences include polymerase chain reactions (PCR) and restriction fragment length polymorphisms (RFLP).
The process for PCR is commonly used to analyze genetic information in many genetic investigations. Even a few molecules of DNA can be amplified to produce large quantities. This property makes PCR ideal for analyzing small samples of DNA. The laboratory procedure involves a cycle of denaturing, annealing, and extending DNA. An increase of temperature triggers denaturing. The hydrogen bonds break between the double stranded helix and they separate. In the process of annealing, the temperature is lowered, which enables primers to attach. Primers are segments of DNA having a free OH group on the 3’ carbon of a nucleotide. These primers align with a very specific sequence of amino acids. After the temperature is increased slightly, a DNA polymerase is able to attach nucleotides to the 3’ carbon of the primer and extend the complementary strand. Each temperature-regulated cycle greatly increases the amount of DNA present, which makes more available for amplification in the next cycle.
For analysis, the PCR product is heated once again and washed over a typing strip. These typing strips are composed of different variations of the alleles amplified during the PCR process. HLA DQa was the first type of strip to be used for forensic analysis. The strip is made by fixing Sequence-Specific Oligonucleotide (SSO) probes to a support. The HLA DQa strip tests for the presence of six different kinds of DQa alleles. There are four main types of alleles fixed to the strip. The 1 allele is then subtyped into 1.1,1.2 and 1.3. The SSOs are placed in nine dots along the strip. The first four probes test for alleles 1-4 respectively. The fifth dot is a control that will attach to any DQa type. Any dot lighter than the control probe is considered invalid. The sixth probe is for the subtype 1.1. The seventh probe will indicate a positive response for 1.2, 1.3 or 4. The eighth detects the presence of subtype 1.3. The last probe will respond to every allele except the 1.3 subtype. These SSO probes grab on to complementary PCR-amplified fragments as they are washed over the strip. The strips are then washed to remove any unattached fragments of DNA. The detection of the DNA on the probe goes back to the primers used during PCR. All of these primers were tagged with biotin. The presence of biotin allows streptavidin to bond to the fragments after they attach to the strip. The streptavidin is chemically linked to horseradish peroxidase (HRP). HRP emits the color blue when in the presence of hydrogen peroxide and tetra-methyl-benzidine (TMB). The resulting strip can then be viewed for analysis. Modern forensics uses an HLA DQA1 testing strip. The advantage of the new strip is the detection of the allele 4 subtypes as well as the allele 1 subtypes.
RFLP comparisons are much more specific tests, but require DNA samples of very high quality to work effectively. The procedures are also much more labor-intensive than PCR. First a restriction endonuclease is used to cut the DNA into millions of fragments. The restriction endonuclease is able to cut a strand when it comes across a specific sequence of nucleotides. These fragments are separated by gel electrophoresis based on their relative sizes. Smaller fragments have less drag in the gel and are carried further by the current. NaOH is then added to the gel to alter the pH and break the duplex DNA molecules into singular strands. The fragments are transferred to a nylon membrane in a process called Southern Blotting. In this procedure the nylon membrane is placed on top of the gel. Absorptive material is then placed on top of the membrane, pulling the gel through the nylon into the material. The DNA is carried in the gel medium to the nylon membrane where it is fixed to the sheet. The membrane is baked to more tightly attach the DNA fragments. To ensure no other DNA molecules can attach to the sheet, protein or detergent is added, which blocks all of the vacant spots where a DNA fragment could attach. Now the only way a DNA molecule can bond to the sheet is if it is complementary to the single stranded DNA already fixed to it. Probe fragments of specific genes are then made radioactive and washed over the membrane. If the complementary segment is present on the membrane, the probe will attach. After washing away the unattached probes, X-ray film or a phosphorimager can then be used to detect the presence of an attached probe and thus the presence of the gene in the sample. The variability in the types and sizes of probes allow RFLP to reveal a large amount of very specific information. The membrane can be cleaned of any probe and retested with another providing an even larger amount of data.
As the O.J. Simpson trial best exemplified, one must not be too quick to judge DNA fingerprinting as a fail proof method of determining truth. In this trial both PCR and RFLP methods of analysis were applied to 45 bloodstains. At the scene of the murder, 8 drops of blood along the walkway and back gate were found to be O.J. Simpson’s. Seven bloodstains in O.J.’s Bronco were found to be Nichole Brown and/or Ronald Goldman’s. Three drops of Nichole Brown’s blood were found on O.J. Simpson’s socks and 11 bloodstains found on the Rockingham glove contained the victims’ DNA. In spite of all of this biochemical data indicating O.J. Simpson as the murderer, the defense was able to convince the jury that genetic evidence is only as reliable as those who are in charge of the tests and a verdict of not guilty was pronounced.
Nevertheless, today’s courts are filling up with appeals on the grounds of new reliable genetic evidence. Many innocent men and women can now be justly returned to their freedom. The number of overturned rulings has even fueled the movement against the death penalty as people repeatedly witness just how many false convictions there are. Cases like those of Ronald Cotton are showing that even an eyewitness many not be as reliable as the witness provided in the genetic fabric of life itself.
The maximum potential for this technology is far from being attained. The days may be approaching when a hair follicle dropped into a computer produces the picture of the guilty party in seconds. Why bother even sending police out to look for them? If that hair follicle has the criminal’s antigen information on it, isn’t it possible to produce a virus that targets that individual's unique HLA profile? Then release the virus and wait at the hospital or morgue for the right symptoms. It sounds drastic, but it may be possible…
In 1985 Ronald Cotton was imprisoned for the rape of Jennifer Thompson. She had identified him from pictures and a line-up as the assailant. Circumstantial evidence stacked up against Cotton making it quite clear that Cotton was guilty. He was imprisoned with no flaw in the judicial system to speak of, except one…he was innocent. After 10 years of imprisonment, biochemical technology allowed the comparison of Cotton’s DNA to that of the rapist’s semen, proving his innocence beyond a shadow of a doubt. The same evidence was then used to rightfully convict Robert Poole, a convict who had mentioned the rape to fellow inmates.Since the early 1980’s DNA "fingerprints" have been used to convict or release possible suspects involved in many crimes. The use of these fingerprints by the FBI’s Forensic Science Systems Unit to form the national DNA registry called, Combined DNA Index System (CODIS), is perhaps one of the greatest assets to criminal investigators to date. It is the goal of the FBI to have the Index operating much like the Automated Fingerprint Index System (AFIS). The Standardization Project has insured that all of the genetic information collected across the United States is in a comparable format. All states have authorized the submission of DNA from violent criminals and sexual offenders to CODIS. Almost any type of biological evidence found at a crime scene may be compared to a suspect, including: blood, semen, saliva, bone, tissue, teeth, and even hair follicles.
he methods for obtaining and comparing the genetic samples of evidence and suspects were originally developed to determine the compatibility of Human Leukocyte Antigens (HLA) in individuals for organ and bone marrow transplants. These antigens are highly specific to an individual and are used by the body’s immune system to determine self from non-self. If a transplant is made without first comparing the compatibility of these antigens, there is a very high likelihood that the donated tissue will be rejected and attacked by the recipient’s immune system. The high amount of specificity allows the regions of DNA encoding for the antigens to be extremely useful in identifying one out of several million people. The commonly used methods for isolating and comparing genetic sequences include polymerase chain reactions (PCR) and restriction fragment length polymorphisms (RFLP).
The process for PCR is commonly used to analyze genetic information in many genetic investigations. Even a few molecules of DNA can be amplified to produce large quantities. This property makes PCR ideal for analyzing small samples of DNA. The laboratory procedure involves a cycle of denaturing, annealing, and extending DNA. An increase of temperature triggers denaturing. The hydrogen bonds break between the double stranded helix and they separate. In the process of annealing, the temperature is lowered, which enables primers to attach. Primers are segments of DNA having a free OH group on the 3’ carbon of a nucleotide. These primers align with a very specific sequence of amino acids. After the temperature is increased slightly, a DNA polymerase is able to attach nucleotides to the 3’ carbon of the primer and extend the complementary strand. Each temperature-regulated cycle greatly increases the amount of DNA present, which makes more available for amplification in the next cycle.
For analysis, the PCR product is heated once again and washed over a typing strip. These typing strips are composed of different variations of the alleles amplified during the PCR process. HLA DQa was the first type of strip to be used for forensic analysis. The strip is made by fixing Sequence-Specific Oligonucleotide (SSO) probes to a support. The HLA DQa strip tests for the presence of six different kinds of DQa alleles. There are four main types of alleles fixed to the strip. The 1 allele is then subtyped into 1.1,1.2 and 1.3. The SSOs are placed in nine dots along the strip. The first four probes test for alleles 1-4 respectively. The fifth dot is a control that will attach to any DQa type. Any dot lighter than the control probe is considered invalid. The sixth probe is for the subtype 1.1. The seventh probe will indicate a positive response for 1.2, 1.3 or 4. The eighth detects the presence of subtype 1.3. The last probe will respond to every allele except the 1.3 subtype. These SSO probes grab on to complementary PCR-amplified fragments as they are washed over the strip. The strips are then washed to remove any unattached fragments of DNA. The detection of the DNA on the probe goes back to the primers used during PCR. All of these primers were tagged with biotin. The presence of biotin allows streptavidin to bond to the fragments after they attach to the strip. The streptavidin is chemically linked to horseradish peroxidase (HRP). HRP emits the color blue when in the presence of hydrogen peroxide and tetra-methyl-benzidine (TMB). The resulting strip can then be viewed for analysis. Modern forensics uses an HLA DQA1 testing strip. The advantage of the new strip is the detection of the allele 4 subtypes as well as the allele 1 subtypes.
RFLP comparisons are much more specific tests, but require DNA samples of very high quality to work effectively. The procedures are also much more labor-intensive than PCR. First a restriction endonuclease is used to cut the DNA into millions of fragments. The restriction endonuclease is able to cut a strand when it comes across a specific sequence of nucleotides. These fragments are separated by gel electrophoresis based on their relative sizes. Smaller fragments have less drag in the gel and are carried further by the current. NaOH is then added to the gel to alter the pH and break the duplex DNA molecules into singular strands. The fragments are transferred to a nylon membrane in a process called Southern Blotting. In this procedure the nylon membrane is placed on top of the gel. Absorptive material is then placed on top of the membrane, pulling the gel through the nylon into the material. The DNA is carried in the gel medium to the nylon membrane where it is fixed to the sheet. The membrane is baked to more tightly attach the DNA fragments. To ensure no other DNA molecules can attach to the sheet, protein or detergent is added, which blocks all of the vacant spots where a DNA fragment could attach. Now the only way a DNA molecule can bond to the sheet is if it is complementary to the single stranded DNA already fixed to it. Probe fragments of specific genes are then made radioactive and washed over the membrane. If the complementary segment is present on the membrane, the probe will attach. After washing away the unattached probes, X-ray film or a phosphorimager can then be used to detect the presence of an attached probe and thus the presence of the gene in the sample. The variability in the types and sizes of probes allow RFLP to reveal a large amount of very specific information. The membrane can be cleaned of any probe and retested with another providing an even larger amount of data.
As the O.J. Simpson trial best exemplified, one must not be too quick to judge DNA fingerprinting as a fail proof method of determining truth. In this trial both PCR and RFLP methods of analysis were applied to 45 bloodstains. At the scene of the murder, 8 drops of blood along the walkway and back gate were found to be O.J. Simpson’s. Seven bloodstains in O.J.’s Bronco were found to be Nichole Brown and/or Ronald Goldman’s. Three drops of Nichole Brown’s blood were found on O.J. Simpson’s socks and 11 bloodstains found on the Rockingham glove contained the victims’ DNA. In spite of all of this biochemical data indicating O.J. Simpson as the murderer, the defense was able to convince the jury that genetic evidence is only as reliable as those who are in charge of the tests and a verdict of not guilty was pronounced.
Nevertheless, today’s courts are filling up with appeals on the grounds of new reliable genetic evidence. Many innocent men and women can now be justly returned to their freedom. The number of overturned rulings has even fueled the movement against the death penalty as people repeatedly witness just how many false convictions there are. Cases like those of Ronald Cotton are showing that even an eyewitness many not be as reliable as the witness provided in the genetic fabric of life itself.
The maximum potential for this technology is far from being attained. The days may be approaching when a hair follicle dropped into a computer produces the picture of the guilty party in seconds. Why bother even sending police out to look for them? If that hair follicle has the criminal’s antigen information on it, isn’t it possible to produce a virus that targets that individual's unique HLA profile? Then release the virus and wait at the hospital or morgue for the right symptoms. It sounds drastic, but it may be possible…
Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing
DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'–3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11–Rad50–Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.
Plasma DNA Is More Reliable than Carcinoembryonic Antigen for Diagnosis of Recurrent Esophageal Cancer
Background
Carcinoembryonic antigen (CEA) and plasma DNA are known to be elevated in patients with esophageal cancer and are higher in patients with disseminated disease. The sensitivity and specificity of these markers in the diagnosis of recurrent esophageal cancer have not been compared.
Study Design
Plasma DNA was measured using polymerase chain reaction in 45 patients with esophageal cancer and 44 asymptomatic volunteers. The 95th percentile (19 ng /mL) in the volunteers was used to define normal. Thirty-nine patients had localized cancer and underwent resection, and six had disseminated disease at operation. Plasma DNA was measured preoperatively in all patients, with serum CEA measured in 31. Plasma DNA was measured sequentially during followup in 21 patients, including 7 who developed recurrence. CEA was measured in 14 of 21 patients who had sequential plasma DNA measured and in 6 of 7 patients with recurrence. CEA levels greater than 5.0 ng/mL were used as cut-off.
Results
Plasma DNA was more sensitive than CEA for detecting unresectable esophageal cancer (100% versus 40%), but it had a lower specificity (22% versus 89%).The positive predictive value (19% versus 40%) and negative predictive value (100% versus 89%) were similar for plasma DNA and serum CEA, respectively.
Plasma DNA was also more sensitive than CEA in detecting recurrent esophageal cancer (100% versus 33%). The specificity and positive predictive values were 100% for both tests, but the negative predictive values were higher for plasma DNA. Plasma DNA rose before there was clinical evidence of recurrence in 67% compared with only 17% for CEA.
Conclusions
Elevated plasma DNA is an extremely reliable indicator of the presence of recurrent disease, and, in the majority of patients, it rises before clinical evidence of recurrence. In contrast, a normal CEA should be interpreted cautiously, because it does not exclude recurrent disease.
Carcinoembryonic antigen (CEA) and plasma DNA are known to be elevated in patients with esophageal cancer and are higher in patients with disseminated disease. The sensitivity and specificity of these markers in the diagnosis of recurrent esophageal cancer have not been compared.
Study Design
Plasma DNA was measured using polymerase chain reaction in 45 patients with esophageal cancer and 44 asymptomatic volunteers. The 95th percentile (19 ng /mL) in the volunteers was used to define normal. Thirty-nine patients had localized cancer and underwent resection, and six had disseminated disease at operation. Plasma DNA was measured preoperatively in all patients, with serum CEA measured in 31. Plasma DNA was measured sequentially during followup in 21 patients, including 7 who developed recurrence. CEA was measured in 14 of 21 patients who had sequential plasma DNA measured and in 6 of 7 patients with recurrence. CEA levels greater than 5.0 ng/mL were used as cut-off.
Results
Plasma DNA was more sensitive than CEA for detecting unresectable esophageal cancer (100% versus 40%), but it had a lower specificity (22% versus 89%).The positive predictive value (19% versus 40%) and negative predictive value (100% versus 89%) were similar for plasma DNA and serum CEA, respectively.
Plasma DNA was also more sensitive than CEA in detecting recurrent esophageal cancer (100% versus 33%). The specificity and positive predictive values were 100% for both tests, but the negative predictive values were higher for plasma DNA. Plasma DNA rose before there was clinical evidence of recurrence in 67% compared with only 17% for CEA.
Conclusions
Elevated plasma DNA is an extremely reliable indicator of the presence of recurrent disease, and, in the majority of patients, it rises before clinical evidence of recurrence. In contrast, a normal CEA should be interpreted cautiously, because it does not exclude recurrent disease.
PlateSelect™ RNAi
PlateSelect™ RNAi are customizable 96-well RNAi duplexes. This format is ideal for researchers who want small amounts of RNAi duplexes in a ready-to-transfect plate format.
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RNAi duplexes in customizable 96-well plates
Select well orientation in 96-well plates and choose from Invitrogen’s Stealth™ or BLOCK-iT™ RNAi duplexes. The RNAi duplexes are provided at 1 nmol/well, and they are synthesized on demand, based on the most-up-to-date bioinformatics to reduce the chances of off-target effects.
Select the sequence and orientation of your duplexes to match your transfection protocol
Minimize off-target effects with on-demand, synthesized duplexes based on the most up-to-date bioinformatics
Order 1 to 96 duplexes per plate in a convenient 1 nmol scale—no minimum order
Choose Stealth™ RNAi chemically modified duplexes or BLOCK-iT™ RNAi duplexes using an easy-to-use interface
#rightRTE blockquote { margin-left: 10px; margin-right: 10px }
#mainRTE blockquote { margin-left: 20px; margin-right: 20px }
RNAi duplexes in customizable 96-well plates
Select well orientation in 96-well plates and choose from Invitrogen’s Stealth™ or BLOCK-iT™ RNAi duplexes. The RNAi duplexes are provided at 1 nmol/well, and they are synthesized on demand, based on the most-up-to-date bioinformatics to reduce the chances of off-target effects.
Select the sequence and orientation of your duplexes to match your transfection protocol
Minimize off-target effects with on-demand, synthesized duplexes based on the most up-to-date bioinformatics
Order 1 to 96 duplexes per plate in a convenient 1 nmol scale—no minimum order
Choose Stealth™ RNAi chemically modified duplexes or BLOCK-iT™ RNAi duplexes using an easy-to-use interface
BLOCK-iT™ Pol II miR RNAi Vector Services
BLOCK-iT? Pol II miR RNAi Vector ServicesDNA based or vector mediated RNA Interference (RNAi) is often used for long-term expression, hard to transfect cell lines or for inducible RNAi. Vector technologies allow you to:
Achieve transient or stable target knockdownPerform RNAi in any cell type – even hard to transfect, primary and non-diving cellsRegulate gene inhibition with inducible RNAi expressionStudy long-term gene knockdown
BLOCK-iT? Pol II miR RNAi vectors combine the benefits of traditional RNAi vectors – stable expression and the ability to use viral delivery – with capabilities for tissue-specific expression and multiple target knockdown from the same transcript. These vectors are designed to express artificial miRNAs that are engineered to have 100% homology to your target gene and result in target cleavage.
Table 1: BLOCK-iT? Pol II miR RNAi Entry VectorsmiR RNAi Entry Vector Advantages pcDNA?6.2- GW/miR Pol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors pcDNA?6.2- GW/EmGFP- miR Co-cistronic EmGFP reporter for easy tracking of miR RNAi expressionPol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors
Once your miR RNAi entry vector is generated, our RNAi services team will transfect it into mammalian cells to perform knockdown studies, chain multiple miR RNAi sequences together to knock down more than one target with the same vector, or transfer the miR RNAi sequence to another destination vector. Regardless of your vector choice, once expressed in a cell, the miR RNAi sequence induces an RNAi response resulting in knockdown of the targeted message.
Subcloning BLOCK-iT? Pol II miR RNAi SequencesThe BLOCK-iT? Pol II miR RNAi Subcloning Service includes a Gateway? BP and subsequent LR recombination reaction to move the miR RNAi sequence from the miR RNAi entry vector into the Gateway? destination vector of your choice. Currently there are many compatible destination vectors to choose from including lentiviral vectors and vectors with tissue specific promoters. Each destination vector has different features and benefits for increased flexibility in your experiments. We’ll help you choose the best destination vector that meets your experimental goals.
BLOCK-iT? Lentivirus ProductionFor many disease models the most desirable cell types to use, such as non-dividing or primary cells, cannot be efficiently transfected. Invitrogen’s lentiviral delivery system solves this problem by offering a powerful alternative to routine transfections. Lentiviral delivery has proven to be successful with a variety of cell types including:
Post mitotic or non-dividing cellsPrimary cellsStem cellsGrowth arrested cellsAnimal models
Once your BLOCK-iT? Pol II miR RNAi sequence is in a lentiviral destination vector, Invitrogen’s virus production team can produce either crude or concentrated lentiviral stocks in a variety of quantities and formats to meet your experimental goals.
BLOCK-iT? Pol II miR RNAi Phenotypic AssaysThe ultimate goal of gene knockdown is to observe changes in phenotype. Invitrogen’s Custom Services will work with you to design and execute a wide variety of phenotypic assays using BLOCK-iT? Pol II miR RNAi vectors.
BLOCK-iT? Pol II miR RNAi Custom ResearchInvitrogen’s team of expert scientists has years of experience working with RNAi and will work with you to design your RNAi experiments to reliably achieve your research goals. For more information on any of the BLOCK-iT? Pol II miR RNAi Custom Services listed here, please contact Invitrogen Custom Services.
Achieve transient or stable target knockdownPerform RNAi in any cell type – even hard to transfect, primary and non-diving cellsRegulate gene inhibition with inducible RNAi expressionStudy long-term gene knockdown
BLOCK-iT? Pol II miR RNAi vectors combine the benefits of traditional RNAi vectors – stable expression and the ability to use viral delivery – with capabilities for tissue-specific expression and multiple target knockdown from the same transcript. These vectors are designed to express artificial miRNAs that are engineered to have 100% homology to your target gene and result in target cleavage.
Table 1: BLOCK-iT? Pol II miR RNAi Entry VectorsmiR RNAi Entry Vector Advantages pcDNA?6.2- GW/miR Pol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors pcDNA?6.2- GW/EmGFP- miR Co-cistronic EmGFP reporter for easy tracking of miR RNAi expressionPol II CMV Promoter for constitutive, transient expressionPoly-cistronic miR RNAi expressionTransfer miR RNAi cassettes into other Gateway? pDEST? vectors including Lentiviral vectors
Once your miR RNAi entry vector is generated, our RNAi services team will transfect it into mammalian cells to perform knockdown studies, chain multiple miR RNAi sequences together to knock down more than one target with the same vector, or transfer the miR RNAi sequence to another destination vector. Regardless of your vector choice, once expressed in a cell, the miR RNAi sequence induces an RNAi response resulting in knockdown of the targeted message.
Subcloning BLOCK-iT? Pol II miR RNAi SequencesThe BLOCK-iT? Pol II miR RNAi Subcloning Service includes a Gateway? BP and subsequent LR recombination reaction to move the miR RNAi sequence from the miR RNAi entry vector into the Gateway? destination vector of your choice. Currently there are many compatible destination vectors to choose from including lentiviral vectors and vectors with tissue specific promoters. Each destination vector has different features and benefits for increased flexibility in your experiments. We’ll help you choose the best destination vector that meets your experimental goals.
BLOCK-iT? Lentivirus ProductionFor many disease models the most desirable cell types to use, such as non-dividing or primary cells, cannot be efficiently transfected. Invitrogen’s lentiviral delivery system solves this problem by offering a powerful alternative to routine transfections. Lentiviral delivery has proven to be successful with a variety of cell types including:
Post mitotic or non-dividing cellsPrimary cellsStem cellsGrowth arrested cellsAnimal models
Once your BLOCK-iT? Pol II miR RNAi sequence is in a lentiviral destination vector, Invitrogen’s virus production team can produce either crude or concentrated lentiviral stocks in a variety of quantities and formats to meet your experimental goals.
BLOCK-iT? Pol II miR RNAi Phenotypic AssaysThe ultimate goal of gene knockdown is to observe changes in phenotype. Invitrogen’s Custom Services will work with you to design and execute a wide variety of phenotypic assays using BLOCK-iT? Pol II miR RNAi vectors.
BLOCK-iT? Pol II miR RNAi Custom ResearchInvitrogen’s team of expert scientists has years of experience working with RNAi and will work with you to design your RNAi experiments to reliably achieve your research goals. For more information on any of the BLOCK-iT? Pol II miR RNAi Custom Services listed here, please contact Invitrogen Custom Services.
Structure of WDR5 bound to mixed lineage Leukemia protein-1 peptide
The Mixed Lineage Leukemia protein-1 (MLL1) catalyzes histone H3 Lysine 4 methylation and is regulated by interaction with WDR5 (WD-repeat protein-5), RbBP5 (Retinoblastoma Binding Protein-5), and the Ash2L (Absent, small, homeotic discs-2-like) oncoprotein. In the accompanying investigation, we describe the identification of a conserved arginine containing motif, called the “Win” or WDR5 interaction motif that is essential for the assembly and H3K4 dimethylation activity of the MLL1 core complex. Here we present a 1.7-? crystal structure of WDR5 bound to a peptide derived from the MLL1 Win motif. Our results show that R3765 of the MLL1 is bound in the same arginine binding pocket on WDR5 that was previously suggested to bind histone H3. Thermodynamic binding experiments show that the MLL1 Win peptide is preferentially recognized by WDR5. These results are consistent with a model in which WDR5 recognizes R3765 of MLL1, which is essential for the assembly and enzymatic activity of the MLL1 core complex.
A conserved arginine containing motif crucial for the assembly and enzymatic activity of the Mixed Lineage Leukemia protein-1 core complex
The Mixed Lineage Leukemia protein-1 (MLL1) belongs to the SET1 family of histone H3 lysine 4 methyltransferases. Recent studies indicate that the catalytic subunits of SET1 family members are regulated by interaction with a conserved core group of proteins that include the WD-repeat protein-5 (WDR5), retinoblastoma binding protein-5 (RbBP5), and the Absent small homeotic-2-like protein (Ash2L). It has been suggested that WDR5 functions to bridge the interactions between the catalytic and regulatory subunits of SET1 family complexes. However, the molecular details of these interactions are unknown. To gain insight into the interactions among these proteins we have determined the biophysical basis for the interaction between the human WDR5 and MLL1. Our studies reveal that WDR5 preferentially recognizes a previously unidentified and conserved arginine containing motif- called the “Win” or WDR5 interaction motif, which is located in the N-SET region of MLL1 and other SET1 family members. Surprisingly, our structural and functional studies show that WDR5 recognizes arginine 3765 of the MLL1 Win motif using the same arginine binding pocket on WDR5 that was previously shown to bind histone H3. We demonstrate that WDR5’s recognition of arginine 3765 of MLL1 is essential for the assembly and enzymatic activity of the MLL1 core complex in vitro.
Molecular Mechanism of Sequence-Directed DNA Loading and Translocation by FtsK
Dimeric circular chromosomes, formed by recombination between monomer sisters, cannot be segregated to daughter cells at cell division. XerCD site-specific recombination at the Escherichia coli dif site converts these dimers to monomers in a reaction that requires the DNA translocase FtsK. Short DNA sequences, KOPS (GGGNAGGG), which are polarized toward dif in the chromosome, direct FtsK translocation. FtsK interacts with KOPS through a C-terminal winged helix domain γ. The crystal structure of three FtsKγ domains bound to 8 bp KOPS DNA demonstrates how three γ domains recognize KOPS. Using covalently linked dimers of FtsK, we infer that three γ domains per hexamer are sufficient to recognize KOPS and load FtsK and subsequently activate recombination at dif. During translocation, FtsK fails to recognize an inverted KOPS sequence. Therefore, we propose that KOPS act solely as a loading site for FtsK, resulting in a unidirectionally oriented hexameric motor upon DNA.
Base Sequence and Higher-Order Structure Induce the Complex Excited-State Dynamics in DNA
The high photostability of DNA is commonly attributed to efficient radiationless electronic relaxation processes. We used femtosecond time-resolved fluorescence spectroscopy to reveal that the ensuing dynamics are strongly dependent on base sequence and are also affected by higher-order structure. Excited electronic state lifetimes in dG-doped d(A)20 single-stranded DNA and dG·dC-doped d(A)20·d(T)20 double-stranded DNA decrease sharply with the substitution of only a few bases. In duplexes containing d(AGA)·d(TCT) or d(AG)·d(TC) repeats, deactivation of the fluorescing states occurs on the subpicosecond time scale, but the excited-state lifetimes increase again in extended d(G) runs. The results point at more complex and molecule-specific photodynamics in native DNA than may be evident in simpler model systems.
HARP Is an ATP-Driven Annealing Helicase
DNA-dependent adenosine triphosphatases (ATPases) participate in a broad range of biological processes including transcription, DNA repair, and chromatin dynamics. Mutations in the HepA-related protein (HARP) ATPase are responsible for Schimke immuno-osseous dysplasia (SIOD), but the function of the protein is unknown. We found that HARP is an ATP-dependent annealing helicase that rewinds single-stranded DNA bubbles that are stably bound by replication protein A. Other related ATPases, including the DNA translocase Rad54, did not exhibit annealing helicase activity. Analysis of mutant HARP proteins suggests that SIOD is caused by a deficiency in annealing helicase activity. Moreover, the pleiotropy of HARP mutations is consistent with the function of HARP as an annealing helicase that acts throughout the genome to oppose the action of DNA-unwinding activities in the nucleus.
Scientists Decode Set of Cancer Genes
For the first time, researchers have decoded all the genes of a person with cancer and found a set of mutations that might have caused the disease or aided its progression.Using cells donated by a woman in her 50s who died of leukemia, the scientists sequenced all the DNA from her cancer cells and compared it to the DNA from her own normal, healthy skin cells. Then they zeroed in on 10 mutations that occurred only in the cancer cells, apparently spurring abnormal growth, preventing the cells from suppressing that growth and enabling them to fight off chemotherapy.The findings will not help patients immediately, but researchers say they could lead to new therapies and would almost certainly help doctors make better choices among existing treatments, based on a more detailed genetic picture of each patient’s cancer. Though the research involved leukemia, the same techniques can also be used to study other cancers.“This is the first of many of these whole cancer genomes to be sequenced,” said Richard K. Wilson, director of the Genome Sequencing Center at Washington University in St. Louis and the senior author of the study. “They’ll give us a whole bunch of clues about what’s going on in the DNA when cancer starts to bloom.”The mutations — genetic mistakes — found in this research were not inborn, but developed later in life, like most mutations that cause cancer. (Only 5 percent to 10 percent of all cancers are thought to be hereditary.)The new research, by looking at the entire genome — all the DNA — and aiming to find all the mutations involved in a particular cancer, differs markedly from earlier studies, which have searched fewer genes. The project, which took months and cost $1 million, was made possible by recent advances in technology that have made it easier and cheaper to analyze hundreds of millions of DNA snippets. The study is being published Thursday in the journal Nature.Dr. Wilson said he hoped that in 5 to 20 years, decoding a patient’s cancer genome would consist of dropping a spot of blood onto a chip that slides into a desktop computer and getting back a report that suggests which drugs will work best.Wilson“That’s personalized genomics, personalized medicine in a box,” he said. “It’s holy grail sort of stuff, but I think it’s not out of the realm of possibility.””Until now, Dr. Wilson said, most work on cancer mutations has focused on just a few hundred genes already suspected of being involved in the disease, not the 20,000 or so genes that make up the full human genome.The older approach is useful, Dr. Wilson said, “but if there are genes mutated that you don’t know about or don’t expect, you’ll miss them.”Indeed, 8 of the 10 mutations his group discovered would not have been found with the more traditional approach.A cancer expert not involved with the study, Dr. Steven Nimer, chief of the hematology service at Memorial Sloan-Kettering Cancer Center, called the research a “tour de force” and the report “a wonderful paper.” Dr. Nimer said the whole-genome approach seemed likely to yield important information about other types of cancer as well as leukemia.“It is supporting evidence for the idea that you can’t just go after the things you know about,” Dr. Nimer said.He added, “It would be nice to have this kind information on every patient we treat.”NimeDr. Nimer also predicted that oncologists would quickly want to start looking for these mutations in their patients or in stored samples from former patients, to see if they could help in predicting the course of the disease or selecting treatments.Nimer,Studying cancer genomes has become a major thrust of research. In the past few years the government has spent $100 million dollars for genome studies in lung and ovarian cancers and glioblastoma multiforme, a type of brain tumor. The person who gave her cells for the study at Washington University became not only the first cancer patient, but also the first woman to have her entire genome decoded. Her information will be available only to scientists and not posted publicly, to protect her privacy and that of her family. The only other complete human genomes open to researchers so far have come from men, two scientists known for ego as well as intellect, who ran decoding projects and chose to bare their own DNA to the world: James D. Watson and J. Craig Venter. Their genomes are available for all to inspect.The woman at Washington University had acute myelogenous leukemia, a fast-growing cancer that affects about 13,000 people a year in the United States and kills 8,800. Its cause is not well understood. Like most cancers, it is thought to begin in a single cell, with a mutation that is not present at birth but that occurs later for some unknown reason. Generally, one mutation is not enough to cause cancer; the disease does not develop until other mutations occur.Most of them are just these random events in the universe that add up to something horrible,” said Dr. Timothy J. Ley, a hematologist at Washington University and the director of the study.The researchers chose to study this disease because it is severe and the treatment has not improved in decades.It’s one of the nastiest forms of leukemia,” Dr. Wilson said. “It’s very aggressive. It affects mostly adults, and there’s really no good treatment for it. A very large fraction of the patients eventually will die from their disease.”WilsonBefore starting treatment, the patient they studied had donated samples of bone marrow and skin, so the researchers could compare her normal skin cells to cancer cells from her bone marrow. Some of the patient’s mutated genes appeared to promote cancer growth. One probably made the cancer drug-resistant by enabling the tumor cells to pump chemotherapy drugs right out of the cell before they could do their work. The other mutated genes seemed to be tumor suppressors, the body’s natural defense against dangerous genetic mistakes.Their job is surveillance,” Dr. Wilson said. “If cells start to do something out of control, these genes are there to shut it down. When we find three or four suppressors inactivated, it’s almost like tumor has systematically started to knock out that surveillance mechanism. That makes it tougher to kill. It gets a little freaky. This is unscientific, but we say, gee, it looks like the tumor has a mind of its own, it knows what genes it has to take out to be successful. It’s amazing.”WilsonTests of 187 other patients with acute myelogenous leukemia found that none had the eight new mutations found in the first patient.。That finding suggests that many genetic detours can lead to the same awful destination, and that many more genomes must be studied, but it does not mean that every patient will need his or her own individual drug, Dr. Wilson said.Wilson“Ultimately, one signal tells the cell to grow, grow, grow,” he said. “There has to be something in common. It’s that commonality we’ll find that will tell us what treatment will be the most powerful.”
DNA testing of family .Experts believe that more harm than good
In the United States, there are now many people are keen on their own DNA testing. This test is very simple, only to spend much of the money will be able to know that they may be suffering from some kind of genetic disease risk, but some medical experts on DNA testing that worried about the trend of the spread.
Huolihaji this year, 51-year-old, her mother is an Alzheimer's disease patients, Holly hopes to know whether a genetic test will also suffer from the same disease. So Holly recently spent 2,500 dollars to buy a home DNA test, the statement indicated that the device can detect 19 kinds of suffering from genetic disease risk.
After testing, Holly found himself suffering from Alzheimer's disease is the possibility of 30%. Holly said she felt a better understanding of their own, and now she intends to resign from the sale of the work to become a nurse. The decision, to some extent, DNA testing is done to help her.
However, DNA testing has led to great controversy. Medicine is a family advocate for DNA testing, simple test equipment that can help people find potential disease threats. But scientists do not agree with this practice, first of all, the reliability of test results aside, it is possible the results of those tests would have a negative impact on sentiment; more importantly, DNA testing is likely to trigger a series of medical Ethical issues, such as DNA may be discriminated against, and so on, must be careful.
Huolihaji this year, 51-year-old, her mother is an Alzheimer's disease patients, Holly hopes to know whether a genetic test will also suffer from the same disease. So Holly recently spent 2,500 dollars to buy a home DNA test, the statement indicated that the device can detect 19 kinds of suffering from genetic disease risk.
After testing, Holly found himself suffering from Alzheimer's disease is the possibility of 30%. Holly said she felt a better understanding of their own, and now she intends to resign from the sale of the work to become a nurse. The decision, to some extent, DNA testing is done to help her.
However, DNA testing has led to great controversy. Medicine is a family advocate for DNA testing, simple test equipment that can help people find potential disease threats. But scientists do not agree with this practice, first of all, the reliability of test results aside, it is possible the results of those tests would have a negative impact on sentiment; more importantly, DNA testing is likely to trigger a series of medical Ethical issues, such as DNA may be discriminated against, and so on, must be careful.
Israel plans Buck Beauchamp developed a laboratory animal DNA labeling technology
Israel plans Buck Beauchamp developed a laboratory animal DNA labeling technology, the use of technology can not only determine the identity of the livestock, but also the variety of meat on the market situation and safety testing.
Timbuktu Beacham laboratory Israel is engaged in a food, water, environment and micro-organisms, such as chemical detection laboratory professionals, the technology is to identify the stolen cattle developed. Researchers at birth in cattle that is collecting DNA samples, based on this genetic information to establish the database. Detection, only a small amount of DNA samples collected through computer analysis, to get the animal species, born and reared in such areas.
In recent years, the public response to the concerns of livestock disease, researchers began to apply the technology to cattle and other livestock health and safety testing. The use of DNA database, researchers can determine whether the cow had been tested by the outbreak of mad cow disease, such as infections, may also distinguish whether a mix of milk in the milk and other livestock; of the Jewish people, the use of technology can also be judged Eat beef with the rules provided for in cleanliness standards.
The laboratory of molecular biology expert caja Aviv, said that, at present, subject to a livestock farm, such as foot-and-mouth epidemic after the attack, the approach taken by the normally all likely to be infected livestock slaughtered all out, it is unnecessary . Use of their research and development of DNA labeling technology, can effectively distinguish between the health of livestock, livestock losses to reduce the practical significance. To consumers, the technology can also help them to obtain relevant information, such as by buying beef from the farm which, in what manner to feed, whether or not genuine, and so on, which is to enhance public confidence in meat products would also be useful.
It is said that the laboratory in the near future plan on the establishment of a nationwide cattle DNA database pilot, if successful, Israel will have to become the world's first national DNA database of cattle.
Timbuktu Beacham laboratory Israel is engaged in a food, water, environment and micro-organisms, such as chemical detection laboratory professionals, the technology is to identify the stolen cattle developed. Researchers at birth in cattle that is collecting DNA samples, based on this genetic information to establish the database. Detection, only a small amount of DNA samples collected through computer analysis, to get the animal species, born and reared in such areas.
In recent years, the public response to the concerns of livestock disease, researchers began to apply the technology to cattle and other livestock health and safety testing. The use of DNA database, researchers can determine whether the cow had been tested by the outbreak of mad cow disease, such as infections, may also distinguish whether a mix of milk in the milk and other livestock; of the Jewish people, the use of technology can also be judged Eat beef with the rules provided for in cleanliness standards.
The laboratory of molecular biology expert caja Aviv, said that, at present, subject to a livestock farm, such as foot-and-mouth epidemic after the attack, the approach taken by the normally all likely to be infected livestock slaughtered all out, it is unnecessary . Use of their research and development of DNA labeling technology, can effectively distinguish between the health of livestock, livestock losses to reduce the practical significance. To consumers, the technology can also help them to obtain relevant information, such as by buying beef from the farm which, in what manner to feed, whether or not genuine, and so on, which is to enhance public confidence in meat products would also be useful.
It is said that the laboratory in the near future plan on the establishment of a nationwide cattle DNA database pilot, if successful, Israel will have to become the world's first national DNA database of cattle.
Stepwise chromatin remodelling by a cascade of transcription initiation of non-coding RNAs
Recent transcriptome analyses using high-density tiling arrays1, 2, 3 and data from large-scale analyses of full-length complementary DNA libraries by the FANTOM3 consortium4, 5 demonstrate that many transcripts are non-coding RNAs (ncRNAs). These transcriptome analyses indicate that many of the non-coding regions, previously thought to be functionally inert, are actually transcriptionally active regions with various features. Furthermore, most relatively large (several kilobases) polyadenylated messenger RNA transcripts are transcribed from regions harbouring little coding potential. However, the function of such ncRNAs is mostly unknown and has been a matter of debate2. Here we show that RNA polymerase II (RNAPII) transcription of ncRNAs is required for chromatin remodelling at the fission yeast Schizosaccharomyces pombe fbp1 + locus during transcriptional activation. The chromatin at fbp1 + is progressively converted to an open configuration, as several species of ncRNAs are transcribed through fbp1 +. This is coupled with the translocation of RNAPII through the region upstream of the eventual fbp1 + transcriptional start site. Insertion of a transcription terminator into this upstream region abolishes both the cascade of transcription of ncRNAs and the progressive chromatin alteration. Our results demonstrate that transcription through the promoter region is required to make DNA sequences accessible to transcriptional activators and to RNAPII.
Chemical differences between DNA & RNA
Both RNA and DNA are composed of repeated units. The repeating units of RNA are ribonucleotide monophosphates and of DNA are 2'-deoxyribonucleotide monophosphates.Both RNA and DNA form long, unbranched polynucleotide chains in which different purine or pyrimidine bases are joined by N-glycosidic bonds to a repeating sugar-phosphate backbone.The chains have a polarity. The sequence of a nucleic acid is customarily read from 5' to 3'. For example the sequence of the RNA molecule is AUGC and of the DNA molecule is ATGCThe base sequence carries the information, i.e. the sequence ATGC has different information that AGCT even though the same bases are involved.Consequences of RNA/DNA chemistryThe DNA backbone is more stable, especially to alkaline conditions. The 2' OH on the RNA forms 2'3'phosphodiester intermediates under basic conditions which breaks down to a mix of 2' and 3' nucleoside monophosphates. Therefore, the RNA polynucleotide is unstable.The 2' deoxyribose allows the sugar to assume a lower energy conformation in the backbone. This helps to increase the stability of DNA polynucleotides. The following link shows 3-D models of the DNA and RNA nucleotides.Cytidine deamination to Uridine can be detected in DNA but not RNA because deamination of Cytidine in DNA leads to Uridine not Thymidine. Uridine bases in DNA are removed by a specific set of DNA repair enzymes and replaced with cytidine bases.
DNA, RNA, and Protein: Life at its simplest
DNA: Deoxyribonucleic acid. The double-stranded chemical instruction manual for everything a plant or animal does: grow, divide, even when and how to die. Very stable, has error detection and repair mechanisms. Stays in the cell nucleus. Can make good copies of itself.RNA: Ribonucleic acid. Single-stranded where DNA is double-stranded, messenger RNA carries single pages of instructions out of the nucleus to places they're needed throughout the cell. No error detection or repair; makes flawed copies of itself. Evolves ten times faster than DNA. Transfer RNA helps translate the mRNA message into chains of amino acids in the ribosomes.[Diagram of RNA vs. DNA: chemical structure and composition]Base: a building block of DNA and RNA. There are five different bases: Adenine, Thymine, Guanine, Cytosine, and Uracil (which is found only in RNA and replaces Thymine in DNA).Ribosomes: Message centers throughout the cell where the information from DNA arrives in the form of messenger RNA. The RNA message gets translated into a form the ribosome can understand and tells it which protein building blocks it needs and in what order to assemble them. Ribosomal RNA helps the translation go smoothly. Amino acids: Polypeptide (protein) building blocks.Polypeptides: chains of amino acids. Proteins are made up of several or many polypeptides.Proteins: Chemicals that make up cell and organ structure and carry out reactions throughout the body, from breaking down food to fighting off disease. --------------------------------------------------------------------------------DNA is transcribed into mRNA which is translated into amino acids.This is (diagram source)Everything you ever wanted to know about DNA, RNA, and proteins
ABI 310 sequencer DNA-sequencing of the principles and rules
DNA sequencing at manual and automatic sequencing sequencing, including the Sanger sequencing hand-dideoxy chain termination method and Maxam-Gilbert chemical degradation. In fact automated sequencing of DNA has become the mainstream of sequence analysis. U.S. PE ABI has produced 373-377-310-3700 and 3100, such as DNA-sequencing device, which is a 310-clinical testing laboratories used in most models. This experiment is described in the ABI PRISM 310-DNA sequencer sequencing of the principles and rules. 】 【Principle ABI PRISM 310-gene analysis (that is, DNA sequencer), using capillary electrophoresis to replace traditional flat-polyacrylamide gel electrophoresis, the company's patent application of the four-color fluorescent dyes labeled ddNTP (marking the termination method), through the Single-primer sequencing of PCR reaction, PCR product generated is the difference between a base of 3'''''''' for the end of the 4 different fluorescent dye mixture of single-stranded DNA, making four fluorescent dyes sequencing of PCR Can be a product of a capillary electrophoresis, thus avoiding the inter-lane mobility differences, greatly enhanced the accuracy of the sequencing. Due to the size of the different elements in the capillary electrophoresis mobility is also different from when reading through the capillary window above, the laser detector in the window CCD (charge-coupled device) camera detector of fluorescent molecules can be tested one by one, Excited fluorescence spectrometry by the grating in order to distinguish between different base of information on behalf of the different colors of fluorescent, and CCD imaging cameras simultaneously, the software will automatically change to a different fluorescent DNA sequence, so as to achieve the purpose of DNA sequencing. The results can gel electrophoresis, fluorescence peaks base map or order forms, such as the output. It is automatically a plastic irrigation, automatic injection, automatic data collection, analysis, computer-controlled automatic determination of the sequence of base pairs of DNA fragments, or the size of the quantitative and high-end precision instruments. PE also offers gel polymers, including DNA sequencing gel (POP 6) and GeneScan plastic (POP 4). These gel particles uniform size, equipped with plastic to avoid the inconsistency of the conditions of sequencing accuracy. It mainly by capillary electrophoresis device, Macintosh computers, color printers, such as electrophoresis and the composition of the annex. Computers, including data collection, analysis and operation of equipment such as software. It uses the latest CCD camera detector, so that the DNA sequence has been shortened to 2.5h, PCR fragment size analysis and quantitative analysis for 10 ~ 40min. As the DNA sequencing instruments have, PCR fragment size analysis and quantitative analysis functions, it can be carried out DNA sequencing, heterozygote analysis, single strand conformation polymorphism (SSCP), microsatellite analysis, long fragment PCR, RT -PCR (quantitative PCR) analysis, and so on, in addition to clinical routine DNA sequencing, can also carry out single nucleotide polymorphisms (SNP) analysis, gene mutations, HLA typing, on the forensic identification of individual and family , Micro-organisms and viruses such as typing and identification. 【Reagents and equipment】 1. BigDye sequencing reaction reagent kit is the main BigDye Mix, containing PE Patent four-color fluorescent ddNTP and the general dNTP, AmpliTaq DNA polymerase FS, reaction buffer, and so on. 2. pGEM-3Zf (+) double-stranded DNA templates were 0.2g / L, matching reagent kit. 3. M13 (-21) primer TGTAAAACGACGGCCAGT, 3.2μmol / L, that is, 3.2pmol/μl, matching reagent kit. 4. DNA sequencing template PCR can be a product of single-stranded DNA and the DNA plasmid, and so on. Template concentration should be adjusted in the PCR reaction volume of 1μl get better. Experimental determination of the plasmid DNA, concentration of 0.2g / L, that is, 200ng/μl. 5. Primer On the basis of the determination to take the DNA fragments are designed or reverse primer, preparation 3.2μmol / L, that is, 3.2pmol/μl. If the recombinant plasmid containing universal primer sequences can also be used universal primer, such as the M13 (-21) primer, T7 primers, and so on. 6. Sterilization deionized water or distilled water three. 7.0.2ml or PCR tubes and 0.5ml of isolation cover, PE products. 8.3mol / L sodium acetate (pH5.2) that take 40.8g NaAc? 3H2O dissolved in 70ml distilled water, glacial acetic acid pH adjusted to 5.2, the volume to 100ml, hours after the high-pressure sterilization equipment. 9.70 percent ethanol and ethanol. 10. NaAc / ethanol mixture of ethanol and 37.5ml take 2.5ml 3mol / L NaAc blending, to save room temperature for 1 year. 11. POP 6 sequencing ABI plastic products. 12. Template suppression reagent (TSR) ABI products. 13.10 × electrophoresis buffer ABI products. 14. ABI PRISM 310 automatic DNA sequencer. 15.2400-9600 or PCR-based instrument. 16. Frozen high-speed desktop centrifuge. 17. High-speed desktop centrifuge or centrifuges pocket. Key steps】 1. Sequencing of PCR reaction (1) 0.2ml take control of the PCR, marker number tube will be inserted in the ice particles in the table below plus reagent: The increase in the standard template reagent measured in the control tube BigDye Mix 1μl 1μl The test plasmid DNA 1μl -- pGEM-3Zf (+) double-stranded DNA - 1μl DNA test positive primer 1μl -- M13 (-21) primer - 1μl Deionized water sterilization 2μl 2μl The total volume of reaction 5μl, no light mineral oil or paraffin oil, Gaijin PCR tube, pipe bombs mixing fingers, slightly centrifuge. (2) PCR tube placed in 9600 or 2400 based on PCR amplification instrument. 98 ℃ degeneration 2min after the PCR cycle, PCR parameters to Circulating 96 ℃ 10s, 50 ℃ 5s, 60 ℃ 4min, 25 cycles, amplified after the end of the heat setting 4 ℃. 2. Sodium acetate / ethanol purified PCR product (1) centrifuge mixture, amplified products will be transferred to 1.5ml EP tube. (2) by adding 25μl sodium acetate / ethanol mixture, full oscillation, home ice 10min to precipitate DNA. 12 000r/min at 4 ℃ centrifuge 30 min, the supernatant discarded carefully. (3) plus 70% (V / V) ethanol washing 50μl precipitation 2. 12 000r/min at 4 ℃ centrifuge 5min, care of abandoned liquid supernatants wall and beads, vacuum drying precipitation 10 ~ 15min. 3. Before electrophoresis sequencing of the PCR product of the deal. (1) by adding 12μl of the TSR in centrifuge tubes, severe vibration, to dissolve the full DNA precipitation, centrifugal slightly. (2) solution will be to build the separation 0.2ml PCR tube, slightly centrifuge. (3) in the PCR instrument on thermal denaturation (95 ℃ 2min), in ice cold at first, to be on the plane. 4. Operation on Manual operation of equipment by capillary installed, the location of the capillary correction, manually artificial plastic irrigation operation and the establishment of the sequencing of the order paper. The device will automatically filling plastic to capillary, 1.2kV electrophoresis pre-5min, according to the order of auto-injection program, and then pre-electrophoresis (1.2kV, 20min), the 7.5kV electrophoresis under 2h. After the electrophoresis apparatus will be self-cleaning, filling plastic, into the next sample, and pre-PAGE electrophoresis. Each sample of the total time for electrophoresis 2.5h. After the electrophoresis apparatus will automatically print out the color analysis or sequencing map. 5. The device will automatically sequence analysis, and based on user requirements sequence comparison. If the sequence of known sequence, the sequence can be compared with an asterisk marked difference base, and enhance efficiency. 6. Completed by sequencing equipment and cleaning procedures for equipment maintenance. By calculating】 Sequencing reactions accuracy formula: 100% - difference in the number base (N does not include the number) / 650 × 100% That is, differences in the determination of the base sequence of DNA known standard DNA sequence comparison of different bases, N for the equipment can not read the base. 【Notes and Evaluation】 1. ABI PRISM 310 genetic analyzer is a high-end precision instruments, to be special operations, management and maintenance. 2. In this study sequencing of the PCR reaction of the total volume was 5μl, and not covered by mineral oil, the PCR tube covered sealing is very important, with the exception of Canada finished after Gaijin PCR reagent tube cover, the best selection of the company's PE tube PCR. If the PCR after the end of the PCR solution is less than 4 ~ 4.5μl, the PCR reaction is likely to fail, do not have to carry on and the kind of purification. 3. Sequencing as users only need to provide good purification of DNA samples and primers, a sequence of the PCR reaction using the template, the need for DNA will be different amount, PCR sequencing of the required template low, generally a product of PCR To be 30 ~ 90ng, single-stranded DNA to be 50 ~ 100ng, double-stranded DNA to be 200 ~ 500ng, DNA purity A260nm/A280nm generally 1.6 to 2.0, the best deionized water or distilled water three dissolved DNA, do not have to TE buffer Liquid solution. Primer deionized water or distilled water three 3.2pmol/μl a good match. 4. Sequencing of Experimental use of this kit is a fluorescent BigDye termination of the substrate cycle sequencing kit, a general DNA test can be about 650bp in length. Of the DNA sequencing machines for accuracy (98.5 ± 0.5) % Identified instruments can not read the base N <2%, the required determination of the length of more than 650bp, need to design another primer. In order to ensure a more accurate sequencing can be designed reverse primer on the same template for sequencing, and confirmed with each other. N for the base can be checked manually, in some cases can be read out. In order to enhance the accuracy of sequencing, according to the location of the star tips, analysis of the artificial color map of the premises, the Department of the base for further checking. Sanger dideoxy sequencing principle The need for DNA replication: DNA polymerase, DNA single-strand template, with a 3'-OH at the end of the single-stranded oligonucleotide primers, 4 dNTP (dATP, dGTP, dTTP and dCTP). Using polymerase template for guidance, constantly dNTP will be added to the primer 3'-OH at the end, so that the primer extension, the synthesis of new complementary DNA strand. If a particular nucleotide, double-nucleoside triphosphate (ddNTP), as a result of deoxyribose in the 3 'position of a lack of hydroxy can not be formed with the follow-up to the phosphodiester dNTP key. For example, there is ddCTP, dCTP and the other three dNTP (one mark for the α-32P), to primers, DNA polymerase template and insulation together, can form a whole has the same primer 5'- Side and ddC residues for the 3 'end at the end of a series of fragments of different lengths of the mixture. By denaturing polyacrylamide gel electrophoresis separation obtained from the radioactive zone map for developing a new synthesis of different length of the DNA chain of distribution of C to provide accurate information, so as to the location of C will be determined. A similar approach in ddATP, ddGTP and ddTTP existing conditions, can be obtained at the same time were ddA, ddG and ddT residue for the 3 'end of the head of the three short fragments of varying. The system will be a mixture of four parallel increase in the place of denaturing polyacrylamide gel electrophoresis gel electrophoresis board for each product in each of the components according to their length will be different from the separation, obtained from the radioactive Map image. Map can be obtained directly from the reading of the DNA base sequences.
The magic of DNA technology
Old saying, "Long Long-sheng, Feng Feng-sheng, the son of a rat holes will be." All the biological characteristics of DNA molecular structure by the decision. Biological differences between species, due to the genome sequence differences in rank due to differences in species, is due to the genome structure of the gene expression differences. In short, is the number of nucleotide differences in the number of permutation and combination and resulted in different species. Now shows that only 0.1 percent among people% of the genetic differences between chimpanzees and human genes, only 1%% of the difference. The use of the characteristics of the fight against crime, mainly: on-site analysis can be extracted from animals, plants and micro-organisms as well as the DNA, to determine the scope of the investigation, that tracking suspects. 1985 British biologist at the University of Leicester in the study, Professor Li克杰弗里斯gene variant gene was found on some small structures, which are sufficient to distinguish between the different structure of the individual. As a result, he thought of the possibility of the use of the structural differences to distinguish between different people, and to draw the world's first piece of DNA fingerprinting. The DNA identification, the police around the world attach great importance to its traditional biological forensic examination can only rule out suspects, not identified a suspect technology to a new stage. At present, the technology has been most countries in the world available. For more than 20 years, DNA identification techniques have been establishing a DNA fingerprint, PCR amplification and RFLP analysis of mitochondrial DNA sequencing and other major technology. These new technologies and new ways to test genetic markers of increasing the information provided by the ever-increasing, so that the test toward fast, sensitive, accurate and trace, automation and quality requirements of the samples are becoming less and less development, more Good to meet the various needs of the criminal investigation, and so on. DNA fingerprinting And even the biological diversity of the human individual, in essence, is that the molecular structure of DNA differences. This use of different technicians using autoradiography and other advanced analytical tools can be drawn as a specific commodity, like the bar-code DNA map. This pattern of the individual as between people of different, like fingerprints, are highly specific and therefore referred to as DNA fingerprinting. DNA fingerprinting of the band is based on the law of the genetic parents of the transfer, in the absence of mutation, the genetic offspring of the half from the father and the other half from the mother, can not be with both parents do not have the genetic markers, son DNA fingerprinting on behalf of all the bands in all of its biological parents of DNA fingerprinting to find the appropriate location. However, DNA fingerprints are also required larger samples are not suitable for the old, corrupt, and other samples to identify the limitations, since the 1990s, the establishment of PCR technology, DNA fingerprinting techniques to reduce the use of gradually. PCR technology PCR polymerase chain reaction is the acronym in English (PolymeraseChainReaction, referred to as PCR). In short, PCR is specific to the use of DNA polymerase genes do in vitro or test-tube with a large number of synthesis. It will be a limited number of DNA amplification of DNA fragments of the astronomical, can trace, old and the degradation of biological samples to do DNA analysis. At present, the technology used, for some genes can be copied to the original 10000-100000 million times. As a result, both in paleontology fossils, remains of historical figures, or a few decades ago in the homicide murderer left behind by the hair, skin or blood, as long as they can to isolate a bit of DNA, will be able to use the PCR amplification to be carried out over Right. This is also the "trace evidence" of the power lies. Forensic PCR technology, the main features are: high sensitivity for trace, corruption and the old samples to identify, specific, simple, short-cycle testing, equipment not ask for much, easy-to-promotion. Mitochondrial DNA sequencing technology Mitochondria also contain DNA, mitochondrial DNA is located in the cytoplasm, with the genetic cytoplasm and at the same time it is maternally inherited. Mitochondrial DNA testing law, that is, for single-parent matriarchal paternity testing to identify the individual, and so on. A typical example is the U.S. Army DNA Identification Laboratory wars of the remains of soldiers killed in the identification of personal identification. The discovery of DNA genetic markers before the forensic science major blood group serology using genetic markers, that is, the traditional blood type. DNA testing technology and a number of traditional criminal techniques, great superiority of the main problems: In many cases, despite trying to cover up criminal suspects, traces of the damage, physical evidence, but the victim's resistance, its own tension and Omissions and other circumstances, it is difficult to avoid leaving traces of blood, hair, body fluids and so on will be made available for DNA testing of biological samples, and the small amount of outdated, as well as the corruption of the old samples can be identified for DNA testing technology in the That crime across time and space to show off their capabilities, play a unique role in providing the material foundation and prerequisite. Gene technology in the police department, and other applications, as Correctional Fue, Xiongwan grams of the sword, so that repeatedly violate the law by all kinds of criminals, but also complicated and confusing case, so as to the true colors of the incident.
About DNA
Deoxyribonucleic acid, or DNA, is a nucleic acid molecule that contains the genetic instructions used in the development and functioning of all known living organisms. The main role of DNA is the lon-term storage of information and it is often compared to a set of blueprints, since DNA contains the instructions needed to construct other components of cells, such as proteins and RNA molecules. The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in regulating the use of this genetic information.
DNA characteristics
a. DNA is DNA from the polymerization of monomer polymer. b. DNA called single-nucleotide of each ODN composed of three parts: a member of nitrogenous base + member of the five carbon sugar (deoxyribose) + member of the phosphate, DNA from C, H, O, N, P composed of five elements. c. DNA containing the base can be divided into four categories: G (Guanine), thymine (Thymine), adenine (Adenine), cytosine (Cytosine) d. DNA base pairs of four nitrogen-containing species-specific composition. That is, four nitrogen-containing bases in the proportion of species with different individuals is the same, but different species, there are differences. e. DNA of four nitrogen-containing bases with a ratio of strange regularity, each in the DNA of organisms A (adenine ODN) = T (thymine ODN) C (cytosine nucleoside - Acid) = G (guanine-nucleotide). A and T hydrogen bonds between the two connected, C and G-linked bond between the three.
The secrets of DNA
When the gene and found that the correlation between the DNA, people still want to know how the DNA is a kind of thing, it is no concrete way of life to so many messages to the new successor of it? First of all, people want to know what DNA is composed of human love is always asked at the end of this plane. The results have called the Levin scientists through research, found that DNA from four of the smaller things, the four things named nucleotide of the total, as the four brothers, all of which nucleotide surname, But the name is different, namely, adenine (A), guanine (G), cytosine (C) and thymine (T), it is difficult to remember the names of the four, but keep in mind that as long as the DNA from four Nucleotide just get together and connect them to each other's no law, but in fact not the same nucleotide, and their combination with each other's ever-changing ways, a great mystery. Now, people basically have to understand the genetic How did it happen. The 20th century, the study found that biology: the body is posed by the cell, the cell membrane, such as the composition of the nucleus and cytoplasm. Known in the nucleus of a material called chromosomes, which is called by some of the DNA (DNA) of the composition of the material. Biological genetic material present in all cells, the substance called nucleic acid. Nucleic acid from the nucleotide polymerization. And each of the nucleotide phosphate, ribose and constitutes a base. There are five bases, respectively, adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). Each contains only five nucleotide base pairs in a. The single nucleotide even as a chain, the two nucleotide chains according to a certain degree of order, and then twisted into a "cannabis"-like, it constitutes a DNA (DNA) of the molecular structure. In this structure, each of the three bases to the genetic composition of a "password" and a DNA base pairs on as many as several million, so each of the DNA is a much of the genetic code, which the possession of genetic information A countless number of such molecules on the DNA present in the chromosomes in the nucleus. As they passed cell division genetic code. Human genetic trait passed by the password. There were about 25,000 genes, each gene is determined by the password. Human gene in both parts of the same, but different. A different part of the decision of the distinction between human beings, that is, the diversity of people. A total of DNA's genetic code 3,000,000,000, the composition of the order of about 25,000 genes.
The structure of dna
DNA is composed of many DNA residue, according to a certain order each other with 3 ', 5'-phosphate ester linked to the composition of long chain. DNA contains a majority of two such long-chain, and some for single-stranded DNA, such as E. coli phage φX174, G4, M13 and so on. Some DNA for the ring, and some for the linear DNA. Contains adenine, guanine, thymine and cytosine base 4. In some types of DNA, 5 - cytosine methylation may to a certain extent replaced by cytosine, wheat germ DNA of the 5 - cytosine-rich in particular, up to 6% of the mole. In some phage, 5 - hydroxymethyl cytosine replaced cytosine. In the late 40, Gabriel Richard (E. Chargaff) found that different species of the base composition of DNA, but the number of adenine equal to the number of its thymine (A = T), guanine cytosine number equal to the number (G = C), and therefore the number of purine and pyrimidine equal to the number of and. Several described by the general level of the structure of DNA. A structure of the primary structure of DNA that is its base sequence. Gene is a fragment of DNA, genetic information stored in its base sequence. In 1975 the United States Gilbert (W. Gilbert) and the United Kingdom's Sanger (F. Sanger) respectively, created the structure of the DNA level, the rapid determination of their total end of 1980 Nobel Prize in Chemistry. Since then, the method also has been improved, many of the primary structure of DNA has been established. If people ring of mitochondrial DNA contains 16,569 base pairs, λ phage DNA contains 48,502 base pairs, rice chloroplast genome contains 134,525 base pairs, tobacco chloroplast genome contains 155,844 base pairs, and so on. Now the United States has plans to 10-15 years in all human DNA molecule of about 3,000,000,000 for the nucleotide sequence out. Secondary structure in 1953, Watson (Watson) and Crick (Crick) put forward the basic structure of DNA fiber is a double helix structure, then this model has been recognized scientists, and to explain to copy, transcription, and other important life processes. After an in-depth study and found that humidity and as a result of base sequences in different conditions, DNA double helix can have a variety of types, mainly divided into A, B and Z three categories. It is generally believed, B cells closest to the structure of DNA conformation, it is very similar to the double helix model. A-DNA and RNA molecules in the two-time transcription, as well as the district Screw the formation of the DNA-RNA hybridization close to the molecular conformation. Z-DNA nucleotide dimer to the left to the wound as a unit, which was the main chain saw (Z)-shaped, and named. This configuration for multi-purine nucleotide chains of alternating pyrimidine area. In 1989, U.S. scientists used scanning tunneling electron microscope to directly observe the double helix DNA double helix DNA ︰ 1952, Austria-American biochemist Chargaff (E.chargaff, 1905 -) was determined by DNA base pairs in the 4 Content and found that methotrexate gland and fat equal to the number of thymine, cytosine and methotrexate fat birds of the same number. This Watson, Crick immediately think of base 4 between 22 corresponds to the relationship between the formation of the gland fat and thymine pair methotrexate, methotrexate fat birds and the concept of cytosine pair. High structure Triplex DNA (T-DNA) In the early 1950s, Wilkins According to the X-ray diffraction pictures have been envisaged in the DNA chain may be 3, Crick later watson and others have to build some T-DNA model. Has found that the three DNA chain can be divided into two categories, namely, three helix structure of the Chinese Academy of Sciences and in 1990 by Bai Chunli, such as scanning tunneling electron microscope techniques to observe the structure of the three Fabian " Three in the spiral structure of DNA double helix structure formed on the basis of the three chains District 3 chain are homologous or homologous pyrimidine purine that the entire base of the pyrimidine purine or, in accordance with section 3 of the chain of sources , The DNA chain can be divided into three elements and between elements within the two groups, according to the 3 components of the chain as well as the relative positions can be divided into Pu-Pu-Py and Py-Pu-Py-two (Pu-generation Table purine chain, Py pyrimidine on behalf of the chain) is one of the most common Py-Pu-Py type, it's 3 in the chain there are 2 for a normal double helix, 3 pyrimidine chain located in the double helix of the big ditch, Purine with the chain in the same direction and with the double helix structure of the rotation together, the three chains in the base pairs of DNA double helix with the same, that is, their base is still AT, GC matching However, No. 3 chain C must be protonated , And G with only 2 to form hydrogen bonds (normally three hydrogen bonds). Triplex DNA research will help shed further light on the structure of chromosomes and genes in eukaryotic transcription, replication, and re-regulation and control mechanism. In addition three DNA chain have a certain value, such as the availability of single-stranded DNA fragments will be cutting agent (such as the endonuclease) to carry DNA to a specific site, so as to achieve selective chromosome DNA hit off the end, because the cells Transcription factors such as regulation and protein only after the combination of DNA double helix to open its specific gene transcription, transcription factors can not helical and the three combined, it can make use of oligo DNA fragment closure of the transcription factor binding sites in order to reach the turn off harmful genes or Virus genes. Quadruplex DNA Klug and Sundpuist in a simulated 1 kind of spike protozoan telomere DNA of the caterpillar, paragraph 1 of the synthetic DNA sequences and found that under certain conditions, the simulation of G-rich single-stranded DNA to form a quadruplex DNA structure. This chromosome that the end of the telomere single strand between the formation of a four-chain. Kang and others were confirmed by experiments in crystal and in solution, the rich G DNA can form a quadruplex DNA structure. Quadruplex DNA is the basic structure of the G-quadruplex, that is, in the four conjoined at the center there are 4 by a negative charge with the carboxylic oxygen atoms surrounded the "pocket" through the G-quadruplex accumulation can be Elements to form molecules or between right-handed spiral, with the double helix structure of DNA comparison, G-quadruplex spiral 2 significant characteristics: 1, the stability of its decision in the pocket by the combination of cation type, known ion k The combination of quadruple-helix so that the most stable; 2, and its thermodynamic stability of the very nature and dynamics. At present some of the biological DNA sequence analysis that the G-rich DNA sequence found in some of the functions and evolution are very conservative region of the genome, many studies have shown that guanine-rich DNA chain formed by G-DNA may be As the mutual recognition between the molecular components of one of the organisms in the cell plays a special role
Edit this paragraph DNA replication
DNA is the carrier of genetic information, the parental DNA molecule itself to be a template for the accurate reproduction into two copies, and assigned to the two daughter cells go in, the carrier of genetic information to complete its mission. The double-stranded DNA structure to maintain the stability of this type of genetic material and the accuracy of reproduction are extremely important. (A) DNA replication and a half of the Click and Waston made in the DNA double helix structure of DNA to replicate the model during the course of research conducted and found that the process of DNA replication in the base of the hydrogen bonds between the first fracture (through the helicase), the double helix structure of the helicase separating Chain were the template for synthesis of the new chain. As the offspring of each of the DNA chain from a parent and the other is a new synthesis, so-called reservations and a half to copy (semiconservative replication). (B) DNA replication 1. Double helix of DNA helicase (1) single-stranded DNA binding protein (single-stranded DNA binding protein, ssbDNA protein) (2) DNA helicase (DNA helicase) (3) DNA chain solution 2. Okazaki fragments and the semi-discontinuous replication 3. Copy and lead to the termination of the (C) telomeres and telomerase American Indians in 1941 McClintock (Mc Clintock) put on the telomere (telomere) of the hypothesis that there is the inevitable end of the chromosome of a special structure - telomere. Now the role of telomeres of chromosomes are known to have at least two: ① to protect against injury at the end of chromosomes, so that the chromosome remained stable; ② connected with the nuclear lamina, so that the chromosome can be positioning.
Physical and chemical properties of DNA
DNA molecules are polymers, DNA solution for the polymer solution, with very high viscosity. DNA absorption of UV radiation, when the nucleic acid degeneration, the increased absorption value; when the degeneration of nucleic acid can be complex, the value of the absorption will revert to the original level. Temperature, organic solvents, pH, urea, and so on-reagent can be caused by degeneration of DNA molecules, DNA, even if a double bond between the hydrogen bond breaking, double helix to solve. DNA (deoxyribonucleic acid) refers to the DNA (genes and chromosomes an integral part of) the DNA polymer, is the main component of chromosomes. The vast majority of genetic information stored in DNA molecules.
Distribution and function of DNA
Prokaryotic cell chromosome is a long DNA molecule. Eukaryotic cell nucleus in more than one chromosome in each chromosome with only a DNA molecule. But they are generally better than the original nuclear DNA of cells and large molecules and proteins together. The DNA molecule is a function of storing all of the species decided to RNA and protein structure of all the genetic information; planning are the order of biological cells and tissues and synthetic components of time and space; determine the biological life cycle from beginning to end and determine the biological activity of personality. In addition to the chromosomal DNA, a small number of very different structure of the DNA present in the eukaryotic cell's mitochondria and chloroplasts. DNA is the genetic material of the virus DNA.
Discovery of DNA
Since Mendel's laws of genetic re-discovered, it also raises the question: is the genetic material is not an entity? In order to solve the problem of what genes are, the people of nucleic acids and proteins. As early as 1868, people have been found in nucleic acids. Chemists in Germany Hoppe Sailor's lab, a graduate of the Swiss(1844 - 1895), his lab near a hospital threw the bandage with blood of the very sense of Interest, because he knows that those blood to defend human health, germs and "combat" and was killed in action and kill white blood cells of the human body "remains." He carefully put a bandage on the blood collected and used for decomposition of pepsin, and found the bodies of most of the cells break down, but on the nuclear non-functional. He further on the nuclear material within an analysis and found that the nucleus contains a phosphorus and nitrogen-rich material. Hoppe sail with yeast experiments to prove that the material inside the nucleus of the Mitchell findings are correct. He would like to give this separate from the nuclear material is known as "nuclide", was later found that it was acidic, so to be called "nucleic acid." Since then, people have carried out a series of effective nucleic acid. In the early 20th century, Germany Ke Saier (1853 - 1927) and two of his students Jones (1865 - 1935) and Levin (1869 - 1940) study, understand the basic chemical structure of nucleic acids, it Is composed of a number of nucleotide molecules. By nucleotide base pairs, consisting of phosphate and ribose. There are 4 kinds of bases in which (Yin cast a glance gland, Yin guanine, thymine and cytosine), there are two ribose (ribose, deoxyribose), the nucleic acids into RNA (RNA) and deoxyribonucleic acid (DNA) . Levin made his rush to the results of research, mistakenly believe that 4 in the nucleic acid bases in the volume is equal, so as to derive the basic structure of nucleic acids by 4 with different nucleotide bases to connect into a four-nucleoside Acid as a basis for nucleic acid into a polymer, a "four-nucleotide hypothesis." This hypothesis wrong, understanding of the complex structure of nucleic acids from the considerable obstacles, to a certain extent affected the people's understanding of the functions of nucleic acids. It is believed that although the nucleic acid present in the structure of the important - nuclear, but its structure is too simple, it is hard to imagine in the process of genetic What role. Protein nucleic acid than found as early as 30 years, has developed rapidly. Of the 20th century, composed of 20 amino acid protein has been found to be 12, 1940, the whole was found. In 1902, a German chemist Xie Erti charges between amino acid peptide chain link and the theory of the formation of proteins, in 1917, he was synthesized by the glycine-15 leucine and 3 of the 18 components of long-chain . As a result, some scientists the idea, is likely to be genetic in the protein plays a major role. If the nucleic acids involved in genetic, protein and must be linked to the role of the protein. As a result, at that time generally tend to think that biological protein is the carrier of genetic information. In 1928, American scientists Griffith (1877 - 1941) with a capsule, and a strong toxic-free capsule, low toxicity of the pneumococcus experiments on rats. To have He pods high temperature to kill bacteria with no along with pods of live bacteria were injected mice, he found that the incidence of death soon rats, mice at the same time he's in the blood of the isolated bacteria living there pod. This shows that even without passing bacteria from the dead bacteria in the pod have access to what material to make a risk-free bacteria into bacteria have a pod. Assuming that the right thing to do? Griffith in a test tube experiment and found the dead bacteria with the United States are living without passing on the bacteria test tube train at the same time, without passing all the bacteria have become a pod bacteria and found so that no bacteria long pod A protein of the scare is there a risk of dead bacteria in the shell left over from the nucleic acid (as in heating, the pod of nucleic acid has not been damaged). Griffith said the nucleic acid as a "conversion factor." In 1944, the United States Avery bacteriologist (1877 - 1955) from the United States and bacteria have been isolated activity of the "conversion factor", and that such material did a test of the existence of protein test, the results were negative, and to prove "Conversion factor" is the DNA. But this has not been found in a wide range of recognition, it is not suspected at the time of the technology in addition to net protein, the protein residues into play. German-American scientists Delbruck (1906 - 1981) of phage group has a firm belief that Avery's discovery. Because they observed under the electron microscope of the phage into the shape and growth of E. coli. Phage bacterial cell is a host for the virus, individual small, with only the electron microscope to see it. It is like a small tadpole, the external components of the protein by the end of the first film and the sheath, the head of the internal contain DNA, the end of the end of silk sheath there, the substrate and the small hook. When the phage infection of E. coli, the tail end of the first bar in the bacterial cell membrane, and then it will be all the body's DNA were injected into bacteria cells, the protein shell remain in the bacterial cell outside, not from what the role of the . After the bacteria enter the cells of the phage DNA, on the use of bacterial material and rapid synthesis of the phage DNA and proteins, so many copy of the original size of the display exactly the same shape of a new phage until the bacteria were completely disintegrated, leaving only those phage dead bacteria , And then infect other bacteria. In 1952, key members of the phage group Hershey (a 1908) and his students use the Chase advanced isotope labeling, so the E. coli bacteriophage experimental infection. He coli T2 phage DNA markers on 32P, the protein shell markings on 35S. Marking the first use of phage T2 E. coli infection, and then be separated from the results of the phage will be marked with 35S shell out to stay in E. coli, only the internal display with a 32P labeled nucleic acid were injected all the E. coli and E. coli within Phage successful breeding. The DNA test to prove there is transmission of genetic information of the functions of proteins and DNA by the synthesis of the directive. The result was immediately accepted by the academic community. Almost at the same time, Austria biochemist Chargaff (1905 -) of the nucleic acid bases in 4 of the content of the outcome of the re-determination has been made. Avery in the work, if he thought the different species is due to the different DNA, the DNA structure must be very complicated, or difficult to adapt to biological diversity. As a result, he was out of the text "four nucleotide hypothesis," have had a doubt. In the 1948 - 1952 4 years, he made use of Levin times more than the precision of paper chromatography separation of 4 base pairs, with UV absorption spectra to do quantitative analysis, after repeated the experiment many times, and finally arrive at a different Levine. The results show that DNA molecules in purine and pyrimidine equal to the total number of elements, of which purine A gland and an equal number of T thymine, guanine and cytosine G Yin C equal to the number. Description of the DNA molecule A base with T, G and C is the existence of the match, which negates the "four nucleotide hypothesis", as well as to explore the molecular structure of DNA provides an important clue and based on. April 25, 1953, the United Kingdom's "Nature" magazine published in the United States of Watson and Crick of the British University of Cambridge in cooperation with the results of research: DNA double helix model of the molecule, known as the outcome of the later 20 Century biology's greatest discoveries marked the birth of molecular biology. Watson (1928 A) in middle school is an extremely intelligent children, 15-year-old when they entered the study at the University of Chicago. At that time, because of a person to allow an earlier study of the experimental sex education programs so that Watson had the opportunity to complete all aspects from the study of biological science courses. At the university, although Watson in genetics have little formal training, but since reading the Schrodinger's "What is life? - The physical appearance of living cells, "which prompted him to" find the genetic secrets. " He was good at brainstorming to win many long, good at using other people's ideas to enrich themselves. As long as there are convenient, do not have to force yourself to a whole new field of study, can be required knowledge. 22-year-old Watson made a doctorate, and then was sent to Europe to pursue post-doctoral researcher. In order to fully understand a gene's chemical structure of the virus, he went to study chemical laboratory in Copenhagen, Denmark. On one occasion he went to Naples, Italy, tutor to take part in a meeting of biological macromolecules, have the opportunity to listen to the British biologist physical Wilkins (1916 -) speech, Wilkins saw the DNAX-ray diffraction photograph. Since then, to find the key to unlock the structure of DNA in Watson's idea in the minds of the return. What can learn X-ray diffraction analysis of this map? So he went to Britain to study at the University of Cambridge Cavendish Laboratory, during which Watson understanding of the creek. Crick (1916-2004) when high school passion for science, and in 1937 graduated from the University of London. In 1946, he read "What is life? - The physical appearance of living cells ", is determined to use the knowledge of physics to the study of biology, from biology to the interest. In 1947 he re-started the post-graduate study, in 1949, he Perutz used in conjunction with the X-ray study of the technical structure of the protein molecule, so this has been met with Watson. Watson, Crick was more than 12-year-old major, has not yet obtained a doctoral degree. However, very speculative to talk about them, Watson here are actually able to find how a protein is more important than DNA, is Sanshengyouxing. At the same time, Watson was in contact with his people, the creek is one of the most intelligent. They talked every day for at least a few hours to discuss academic issues. The two were complementary with each other to criticize each other, as well as stimulate each other's inspiration. They do not think the answer is to open the molecular structure of DNA genetic key to the mystery. With only the precise X-ray diffraction data in order to more quickly identify the structure of DNA. In order to get DNAX-ray diffraction, Crick invited Wilkins to come to Cambridge for the weekend. In the conversation Wilkins accepted the spiral structure of DNA point of view, but also his partner Franklin (1920-1958, F), as well as lab scientists also have been thinking very hard with the problem of DNA structure model . From from November 1951 to April 1953 for 18 months, Watson, Crick and Wilkins and Franklin are among several important academic exchanges. In November 1951, after listening to Franklin Watson on the structure of the DNA of a more detailed report, inspired by a certain knowledge of the crystal structure analysis of Watson and Crick realized that in order to quickly establish the structure of DNA model, only Be able to use other people's analysis of the data. They quickly made a three-helical structure of DNA idea. By the end of 1951, they invited Wilkins and Franklin to discuss this model, Franklin pointed out that the water content of their DNA to less than half do so for the first time to set up the model failed. One day, Watson went to King's College laboratory Wilkins, Wilkins Franklin recently beat out a system of "B-" DNA of the X-ray diffraction photograph. Watson saw pictures of them at once exciting, heart rate has accelerated, such as images than ever before to be the "A" is much more simple, as long as a little look at the "B-" X-ray diffraction photograph, and then by a simple calculation, Will be able to determine the number of DNA molecules in the number of nucleotide chains. Please help Crick mathematician calculated results show that the source has attracted Yin Fu. According to their results from the Chargaff and got the two nucleic acid purine and pyrimidine two 22 equal As a result, the formation of the concept of base pairs. They desperately to think of the 4 base sequence, time and again drawing on paper-base structure, playing with the model, repeatedly made the assumption that time and again to overthrow their own assumptions. Watson (left) and there is a creek, according to Watson in the idea of playing with their own model, he shifted base to search for the removal of all kinds of matching. Suddenly, he found two hydrogen bonds linking the gland Yin-fat thymine and went so far as to the hydrogen bond of 3-connected guanine cytosine-Yin of have the same shape, so it boosted the spirit. Because the number of purine and pyrimidine why the number should be exactly the same as the mystery solved. Chargaff all of a sudden it became the law of DNA double helix structure of the inevitable result of the. As a result, how the chain as a template synthesis of another complementary sequence of bases is not hard to imagine the chain. In that case, the skeleton of the two chains must have the opposite direction. After Watson and Crick tension in a row, quickly completed a metal model of DNA assembly. From this model to see, DNA from the two components of nucleotide chains, which along with the central axis of intertwined with each other in the opposite direction, much like a spiral staircase handrails on both sides is more than 2 nucleotide chains P-glycoprotein gene combined with the turn of the skeleton, and the pedal base is right. In the absence of accurate information on X-ray, they dare not conclude that the model is entirely correct. Franklin Wilkins is the next step of the scientific method to predict based on the model of the X-ray diffraction pattern with the experimental data to make a serious comparison. They called once again invited Wilkins. Less than two days of work, Wilkins and Franklin used X-ray analysis of the data confirmed the double helix structure of the model is correct, and has written two experiments at the same time the report published in the UK "Nature" magazine. In 1962, Watson, Crick and Wilkins received the Nobel Prize for Physiology and Medicine, and Franklin died of cancer death in 1958 have not been awarded the prize. In the late 1930s, the Swedish scientists on the DNA to prove it is asymmetrical. After the Second World War, with the electron microscope determination of DNA's molecular diameter of about 2nm. Double helix structure of DNA was discovered, greatly shocked the academic community, inspired by the people's minds. From then on, people immediately in order to carry out genetics as the center of a large number of molecular biology research. First of all, around the base of 4 how to encode permutation and combination can show 20 kinds of amino acids for the Center for Experimental Research. In 1967, were cracking the genetic code, DNA in the genes so as to the molecular level to be a new concept. It shows: gene DNA molecules is actually a fragment, biological control is a trait of the genetic material of the functions and structure of the units units. The unit in a number of nucleotide fragments on the order is not arbitrary, but there are implications of the order of the password. A certain structure of the DNA, can control the structure of the corresponding protein synthesis. Protein is an important component of the composition of the organisms, the organisms are the main characters to reflect the adoption of the protein. As a result, the genetic traits of control through DNA control of protein synthesis to be achieved. On this basis, one after another have had a genetic engineering, enzyme engineering, fermentation engineering, protein engineering, the development of biotechnology is bound to make use of the law for the benefit of mankind. The development of modern biology, the more it will show up to take the lead for the subjects.
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