Single Strand Conformation Polymorphism #denaturation #strand #research #biopharma #bio
Single-strand conformation polymorphism (SSCP), is defined as difference in conformation of single-stranded nucleotide sequences of similar length that is induced by differences in the sequences of certain DNA length under certain experimental conditions. This property let us distinguish between sequences by means of gel electrophoresis that separates fragments on the basis of their different conformations.
PRINCIPLE OF THE TECHNIQUE:
The principle of SSCP analysis relies on the fact that single-stranded DNA has defined conformation. Altered conformation due to any base change in the sequence causes the single-stranded DNA to migrate in a different way than the wild-type DNA under non denaturing electrophoresis conditions. Therefore mutant DNA and wild-type samples display different banding patterns or may result in mobility shift.
· PCR AMPLIFIED DNA
First step for SSCP involves the amplification of our desired DNA by polymerase chain reaction. A reaction mixture is made containing desired DNA, primers, dNTPs, taq polymerase, MgCl2, tris HCL and KCl.
The amplified DNA is mixed with same volume of buffer containing EDTA, xylene cyanol, bromophenol blue and formamide and denatured at high temperature.
· ABRUPT COOLING
Reaction mixture is then immediately transferred to ice which prevents the reannealing of double strands and results in adaptation of certain conformations by single strands of DNA.
· GEL ELECTROPHORESIS
Gel is prepared by using polyacrylamide (acrylamide and bis-acrylamide), TEMED (tetramythylethylenediamine), ammonium persulphate in TBE (tris, boric acid, EDTA. Gel is then poured into electrophoresis tank and allowed to cool. Running buffer is added and denatured DNA sample is loaded.
Several DNA molecules reanneal during gel electrophoresis. For this purpose we have to make dilutions and briefly nature by boiling for short term.
Bands are analyzed on the basis comparison between of their number of bands and mobility shift of normal and experimental DNA. If the number of bands is more, this tells about the heterozygous mutant. While if the number of bands is same in both reference and experimental DNA but there is a mobility shift, it tells about homozygous mutant.
If the mobility shift is observed it tells about the mutation. But if the bands are at same location then the individual is normal
· It enables to detect known as well as unknown mutations in the population
· It can be used for diagnostic purposes
· It can be used to determine the homozygous or heterozygous mutants
· It can be used to determine the variation in virus strains that may be the mutations undergone by the virus for resistance
· It has been widely used for genetics of hypertension such as monogenic as well as polygenic disorders.
· Mutations undetected at certain conditions may get detected on other conditions
· Sensitivity decreases if fragment length is large
· High rate of reannealing of strands is observed during electrophoresis.
· Only detects the presence or absence of mutation. It does not let us know about the type of mutation.