Use TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis.

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1 1 of 6 4/29/2009 2:49 PM Cite as: Cold Spring Harb. Protoc.; 2006; doi: /pdb.prot3815 Protocol Detection of Mutations by Single-strand Conformational Polymorphism Joseph Sambrook and David W. Russell This protocol was adapted from Molecular Cloning, 3rd edition, by Joseph Sambrook and David W. Russell. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001 INTRODUCTION Single-strand conformational polymorphism (SSCP), one of several methods used to scan segments of DNA for mutations, exploits the electrophoretic differences in mobilities between single-stranded mutant and wild-type DNAs. MATERIALS 10x Amplification buffer Include 0.01% (w/v) gelatin in the buffer. 10x TBE electrophoresis buffer Use TBE at a working strength of 1x (89 mm Tris-borate, 2 mm EDTA) for polyacrylamide gel electrophoresis. 6x Gel-loading buffer I Formamide loading buffer Human genomic DNA to be screened for point mutations Dissolve the DNA at 10 µg/ml in TE (ph 7.6). Oligonucleotide primers, forward and reverse (35 µm each) in TE (ph 7.6) Restriction enzymes (optional) Thermostable DNA polymerase Taq DNA polymerase is recommended. [ - 32 P]dCTP (3000 Ci/mmole, 10 mci/ml) dntp solution (PCR grade) containing all four dntps, each at a concentration of 1 mm (ph 7.0) METHOD 1. In a sterile 0.5-ml microcentrifuge tube, mix in the following order: 1 mm dntp solution 1 µl 10x amplification buffer 2 µl 35 µm 5'-oligonucleotide solution 1 µl (35 pmoles) 35 µm 3'-oligonucleotide solution 1 µl

2 2 of 6 4/29/2009 2:49 PM 10 µci/µl [ - 32 P]dCTP 1 µl human genomic DNA thermostable DNA polymerase 10 µl (100 ng) 1-3 units H 2 O to 20 µl [ - 32 P]dCTP is incorporated in the PCRs to label the amplified DNA uniformly. 32 P-labeled oligonucleotide primers can be used in place of [ - 32 P]dCTP to produce an end-labeled DNA. If possible, set up control reactions using two DNA samples known to contain alleles that differ in sequence by one or more base pairs and that are known to resolve on SSCP gels. In addition, set up a contamination control in which no template DNA is added to the reaction. 2. If the thermal cycler is not fitted with a heated lid, overlay the reaction mixtures with 1 drop (approx. 50 µl) of light mineral oil to prevent evaporation of the samples during repeated cycles of heating and cooling. Alternatively, place a bead of paraffin wax into the tube if using a hot start protocol. Place the tubes in the thermal cycler. 3. Amplify the nucleic acids using the denaturation, annealing, and polymerization times and temperatures listed in the table. For advice on thermal cycler programs, please see The Basic Polymerase Chain Reaction. Cycle Number Denaturation Annealing/Polymerization 30 cycles 5-30 sec at 94 C min at 68 C Last cycle 1 min at 94 C 5-10 min at 68 C Times and temperatures may need to be adapted to suit the particular reaction conditions. 4. While the thermal cycler program is running, prepare a 5.5% polyacrylamide gel containing 10% (v/v) glycerol in 1x TBE gel buffer. 10x TBE gel buffer 10 ml 29:1% acrylamide:bisacrylamide solution 18 ml 10% ammonium persulfate 0.5 ml glycerol H 2 O 10 ml 61.5 ml Mix the reagents by gentle swirling or stirring. This volume of gel solution is sufficient for one polyacrylamide gel of standard size (40 x 40-cm plates with 0.4-mm spacers). The volume of the gel solution can be increased or decreased as needed for other gel sizes. Use the same stock of 10x TBE gel buffer to prepare enough 1x TBE gel buffer to fill the tanks of the electrophoresis apparatus. 5. Assemble and tape together two 40 x 40-cm glass electrophoresis plates with 0.4-mm spacers. To obtain maximum resolution of single-stranded DNA conformers, it is important to use thin-gel spacers that are less than or equal to 0.4 mm in thickness. 6. Add 100 µl of TEMED to the gel solution. Mix the solution by gently swirling the flask, and pour the gel.

3 3 of 6 4/29/2009 2:49 PM Work quickly as the acrylamide solution will polymerize rapidly. For instructions on pouring thin gels, please see Preparation of Denaturing Polyacrylamide Gels. 7. Assemble the polymerized gel into an electrophoresis apparatus at room temperature. Fill the buffer tanks with 1x TBE gel buffer made from the same stock as the gel solution. 8. (Optional) If the amplified DNA fragment is to be digested with a restriction enzyme, remove the PCR tubes from the thermal cycler at the end of the run and place them on ice. Set up the following restriction enzyme digestion: PCR solution 5 µl 10x restriction enzyme buffer 4 µl restriction enzyme (2-50 units) 2 µl H 2 O 29 µl Incubate for 1-2 hours at the temperature appropriate for the restriction enzyme. 9. Dilute either 1.5 µl of the original PCR (from Step 3) or 5 µl of the restriction-enzyme-digested PCR (from Step 8) into 20 µl of sucrose gel-loading buffer. Dilute similar aliquots into 20 µl of formamide dye mix. 10. Boil the formamide-containing samples for 6 minutes, and then plunge the tubes directly into ice. 11. Use a Pasteur pipette or a Hamilton syringe to wash out the wells of the polyacrylamide gel with 1x TBE gel buffer. With a drawn-out glass capillary tube or a micropipettor equipped with a gel-loading tip, load 2 µl of each sample on the polyacrylamide gel. 12. Apply 6-7 V/cm (approx. 250 V [and 15 ma] for a 40 x 40-cm gel) to the gel for approx. 14 hours. 13. At the completion of electrophoresis, separate the glass plates, and transfer the gel to a sheet of Whatman 3MM filter paper. Dry the gel on a vacuum dryer for minutes. 14. Subject the dried gel to autoradiography for 4-16 hours at room temperature without an intensifying screen. REFERENCES 1. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: [Medline] 2. Orita, M., Iwahana, H., Kanazawa, H., Hayashi, K., and Sekiya, T Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc. Natl. Acad. Sci. 86: [Abstract/Free Full Text] Caution Formamide Formamide is teratogenic. The vapor is irritating to the eyes, skin, mucous membranes, and upper respiratory tract. It may be harmful by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood when working with concentrated solutions of formamide. Keep working solutions covered as much as possible.

4 4 of 6 4/29/2009 2:49 PM Caution Radioactive substances Radioactive substances: When planning an experiment that involves the use of radioactivity, consider the physicochemical properties of the isotope (half-life, emission type, and energy), the chemical form of the radioactivity, its radioactive concentration (specific activity), total amount, and its chemical concentration. Order and use only as much as needed. Always wear appropriate gloves, lab coat, and safety goggles when handling radioactive material. X rays and gamma rays are electromagnetic waves of very short wavelengths either generated by technical devices or emitted by radioactive materials. They might be emitted isotropically from the source or may be focused into a beam. Their potential dangers depend on the time period of exposure, the intensity experienced, and the wavelengths used. Be aware that appropriate shielding is usually made of lead or other similar material. The thickness of the shielding is determined by the energy(s) of the X rays or gamma rays. Consult the local safety office for further guidance in the appropriate use and disposal of radioactive materials. Always monitor thoroughly after using radioisotopes. A convenient calculator to perform routine radioactivity calculations can be found at: 6x Gel-loading Buffer I 0.25% (w/v) xylene cyanol FF 40% (w/v) sucrose in H 2 O 0.25% (w/v) bromophenol blue Store at 4 C. Amplification Buffer, 10X 500 mm KCl 100 mm Tris-Cl (ph 8.3 at room temperature) 15 mm MgCl 2 Autoclave the 10x buffer for 10 minutes at 15 psi (1.05 kg/cm 2 ) on liquid cycle. Divide the sterile buffer into aliquots and store them at -20 C. Formamide loading buffer 80% (w/v) deionized formamide 1 mg/ml xylene cyanol FF 1 mg/ml bromophenol blue

5 5 of 6 4/29/2009 2:49 PM 10 mm EDTA (ph 8.0) Purchase a distilled deionized preparation of formamide and store in small aliquots under nitrogen at -20 C. TBE buffer Prepare a 5X stock solution in 1 L of H 2 O: 54 g of Tris base 27.5 g of boric acid 20 ml of 0.5 M EDTA (ph 8.0) The 0.5X working solution is 45 mm Tris-borate/1 mm EDTA. TBE is usually made and stored as a 5X or 10X stock solution. The ph of the concentrated stock buffer should be ~8.3. Dilute the concentrated stock buffer just before use and make the gel solution and the electrophoresis buffer from the same concentrated stock solution. Some investigators prefer to use more concentrated stock solutions of TBE (10X as opposed to 5X). However, 5X stock solution is more stable because the solutes do not precipitate during storage. Passing the 5X or 10X buffer stocks through a 0.22-µm filter can prevent or delay formation of precipitates. dntp solution Dissolve each dntp (deoxyribonucleoside triphosphates) in H 2 O at an approximate concentration of 100 mm. Use 0.05 M Tris base and a micropipette to adjust the ph of each of the solutions to 7.0 (use ph paper to check the ph). Dilute an aliquot of the neutralized dntp appropriately, and read the optical density at the wavelengths given in the table below. Calculate the actual concentration of each dntp. Dilute the solutions with H 2 O to a final concentration of 50 mm dntp. Store each separately at -70 C in small aliquots. For polymerase chain reactions (PCRs), adjust the dntp solution to ph 8.0 with 2 N NaOH. Commercially available solutions of PCR-grade dntps require no adjustment. Base wavelength (nm) Extinction Coefficient (E) (M -1 cm -1 ) A x 10 4 G x 10 4 C x 10 3 T x 10 3 For a cuvette with a path length of 1 cm, absorbance = EM. 100 mm stock solutions of each dntp are commercially available (Pharmacia). Copyright 2006 by Cold Spring Harbor Laboratory Press. Online ISSN: Terms of Service All rights reserved. Anyone using the procedures outlined in these protocols does so at their own risk. Cold Spring Harbor Laboratory makes no representations or warranties with respect to the material set forth in these protocols and has no liability in connection with their use. All materials used in these protocols, but not limited to those highlighted with the Warning icon, may be considered hazardous and should be used with caution. For a full listing of cautions, click here.

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