Single Strand Conformation Polymorphism (SSCP) Analysis using Nondenaturing PAGE

Principle

Single-strand conformation polymorphism (SSCP) technique is a simple and efficient means to detect any small alteration in PCR-amplified products. It is based on the assumption that subtle nucleic acid changes affect the migration of single-stranded DNA fragments and, therefore, result in visible mobility shifts across a nondenaturing polyacrylamide gel (Orita et al., 1989). Polyacrylamide gels were used for analysis of DNA with specialized buffer systems and without urea. In nondenaturing PAGE the components used to synthesize matrix were acrylamide monomers, N, N-methylene bisacrylamide (Bis), ammonium persulphate (APS) and N,N,N’,N’-tetramethylenediamine (TEMED).Ammonium persulphate (APS) when dissolved in water generates free radicals, which activate acrylamide monomers inducing them to react with other acrylamide molecules forming long chains. These chains cross-linked with Bis.TEMED acts as catalyst for gel formation because of its ability to exist in free radical form. The acrylamide and bisacrylamide was used in 49:1 ratio using autoclaved HPLC water added to make 100 ml volume. This 49:1 acrylamide-bisacrylamide solution was dissolved completely using magnetic stirrer and kept it in refrigerator till used.
 

Reagents

Preparation of SSCP Gel
    The percentage of SSCP gels used varied from 8-20 percent but most of the primers were optimized with good results in 12 % PAGE solution.

    Composition of 12% nondenaturing PAGE Solution (For 100 ml)

Reagent	Volume
50% Acrylamide-bisacrylamide	24 ml
10 X TBE	05 ml
Glycerol	10 ml
Autoclaved HPLC water	61 ml
Total	100 ml

The gel mixture was kept dissolved completely and stored at -20°C till it was used.
 

Procedure

The PCR-SSCP procedure included following steps viz; PCR amplification of the gene fragments, resolution in nondenaturing PAGE and visualization using silver staining. PCR conditions were optimized for PCR-SSCP by testing a number of variables such as concentration of DNA, Taq polymerase, dNTPs, MgCl2 and temperature profiles. The PCR amplification protocol for all the SSCP primers used were same except the annealing temperature, which varied between primers.

The Single Strand Conformation Polymorphism analysis of amplified gene fragments was carried out using Bio-Rad Protein II xi Cell vertical gel electrophoresis unit (Bio-Rad laboratories).

1. The two glass plates were washed thoroughly using tap water with detergent and rinsed initially under running tap water till no remains of detergent were left. The plates were wiped two times with tissue paper soaked in distilled water first, 70 percent alcohol and air dried. The similar through cleaning treatment was given to spacers and comb to ensure proper alignment of 20 cm glass plates.
2. The gel sandwich was assembled on a clean surface laying down the long rectangular plate first, then two spacers of equal thickness along the long edges of plate and the short plate was placed on the rectangular plate. The two glass plates with spacers between them were fitted well with proper alignment by tightening the screws of both two sandwich clamps. The cleaned comb (20 wells) was inserted from the top side of the gel sandwich and immediately applied the clamps over the plates containing comb to create sharp wells.
3. The bottom side of the gel sandwich was sealed using 10 ml of 12% gel mix. The gel sandwich was kept in slanting position and the solution mixed with 50 µl APS and 20 µl TEMED was injected between the two glass plates using syringe with fine tip and allowed to polymerize for 10 minutes.
4. After polymerization the assembled gel sandwich was placed in alignment slot of casting stand. The 12 % native PAGE gel mix (25ml) was prepared by adding APS (100 µl) and TEMED (40 µl) at a time and mixed well. This gel mix was filled from upper side of gel sandwich using syringe smoothly without any bubble and clamps were immediately applied over the comb to ensure sharp wells. The gel was kept undisturbed at least 45 minutes for polymerization.
5. After polymerization the comb was removed and wells were flushed with 0.5x buffer. The gel sandwich was placed in electrophoresis tank with notched plate facing towards the buffer reservoir. The reservoir of the electrophoresis tank was filled with 0.5 x TBE and the gel was given pre-run at 200 volts at constant temperature for minimum 45 minutes. Ice cooled water circulation with electric pump applied to central cooling core of assembly to maintain constant temperature.
6. About 4 µl PCR product and 12 µl of a formamide dye was prepared in PCR tube and denatured at 95°C for 10 minutes in the Biometra PCR machine. After denaturation the samples were immediately kept in ice chilled box and kept in -20°C deepfreeze for 10 minutes.
7. After completion of pre-run the wells were flushed again using buffer. The samples were loaded on a nondenaturing 12-20% acry1amide:bis-acrylamide (49:l) gel with gel loading tip and immediately electrophoresis was performed in 0.5 X Tris borate (pH 8.3)-EDTA buffer at 10-12.5 volts/cm for 2-24 hr at room temperature depending on the optimized conditions for each primer.
8. After completion of the electrophoresis for required time the glass plates were removed from the assembly. There after it is subjected to silver staining to visualize bands.