John M. S. Bartlett.pdf - Bio-Nica.info

330 Han and Robinson
sequence differences between two segments of DNA, there are cases where differences
are difficult to detect. Changing primers may make a difference. It is possible that the
specific fragment will not show migration differences even if there are substitutions
present. Analysis of overlapping fragments of the region of interest is one solution
to circumvent this difficulty.
The techniques required for the SSCP method are not complicated, and the whole
procedure is time saving (only one-round PCR is needed) compared with other methods
for detecting mutations and DNA polymorphisms. Because of its simplicity and
reliability, PCR-SSCP has been used extensively for identifying alleles and genotypes
in both basic and clinical investigations.
2. Materials
1. Thermal cycling (PCR) machine.
2. AmpliTaq ® DNA polymerase, 5 U/µL (stored at –20°C).
3. 10× PCR buffer stored at –20°C): 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM
MgCl 2 .
4. dNTP (1.25 mM) stored at –20°C.
5. Primers diluted to a concentration of 20 µM (see Note 1).
6. [α 32 P]-dCTP (New England Nuclear, Boston, MA, BLU013H, 3000 Ci/mmol).
7. DNA templates (100 ng/µL genomic or 110 diluted cDNA).
8. 0.1% SDS and 10 mM EDTA, pH 8.0.
9. Loading buffer: 10 mM NaOH, 95% formamide, 0.05% bromphenol blue, 0.05% xylene
cyanol.
10. Long Ranger 50% stock gel solution (FMC BioProducts, Rockland, ME; see Note 2).
11. 10× TBE buffer: 0.89 M Tris-borate; 0.02 M EDTA, pH 8.0.
12. TEMED.
13. 10% Ammonium Persulphate (APS, after dissolving in water, store at –20°C).
14. Glycerol (Ultra Pure, Life Technologies, Gaithersburg, MD).
15. Sigmacote (Sigma Chemical Co., St. Louis, MO).
16. Acrylamide gel electrophoresis supplies and equipment (see Note 3).
17. Whatman 3MM chromatography paper.
18. Plastic wrap (such as Saran Wrap).
19. Vacuum gel dryer.
20. Gel cassette with an enhancing screen.
21. X-ray films.
22. Lab safety area for working with radioactive materials (see Note 4).
3. Methods
3.1. PCR Amplification (see Note 5)
The PCR should contain the following ingredients (in µL): 10× PCR Buffer (2.0);
1.25 mM dNTP (3.2); 20 µM 5′ primer (1.0); 20 µM 3′ primer (1.0); 5 U/µL AmpliTaq ®
(0.1); Experimental template (2.0); [ 32 P]-dCTP (0.1); and H 2 O (10.6) for a total
of 20 µL.
PCR amplification is performed for 30 cycles (denaturation at 95°C for 30 s, annealing
at 55°C (see Note 6) for 30 s, extension at 72°C for 90 s) with a final extension at
72°C for 7 min. PCR amplified DNA may be kept at 4°C or stored at –20°C.