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…
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