Single Strand Conformation Polymorphism (SSCP) Analysis using Nondenaturing PAGE
|Author: D.S. Kale and B.R. Yadav|
|Affiliation: Livestock Genome Analysis Laboratory (DCB), National Dairy Research Institute (NDRI), Karnal-132001.Haryana State. India|
|Source: Protocol Online|
|Date Added: Wed Sep 23 2009|
|Date Modified: Fri Sep 25 2009|
|Abstract: This protocol, with brief principle and procedure will be informative and useful for molecular biology researchers working on genetic polymorphism study using SSCP technique by nondenaturing polyacrylamide gel electrophoresis (PAGE)|
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.
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)
|50% Acrylamide-bisacrylamide||24 ml|
|10 X TBE||05 ml|
|Autoclaved HPLC water||61 ml|
The gel mixture was kept dissolved completely and stored at -20°C till it was
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).