Single-stranded Conformation Polymorphism (SSCP) distinguishes DNA molecules even they are of the same size due to the differences in the nucleotide sequences. Small changes in the nucleotide sequences can be detected through conformational changes in the DNA.
SSCP analysis includes the extraction of DNA from environmental samples. PCR is then conducted using fluorescent-labeled forward primer and reverse primer specific for the target gene. Double-stranded DNA converted to Single-stranded DNA through lambda exonuclease which digests the phosphorylated strand.
Steps associated with SSCP are
- DNA Extraction
- PCR Amplification of DNA fragments
- Denaturation of amplicons to SSDNA by exonuclease digestion
- Separation of amplicons using polyacrylamide gel electrophoresis
- Visualization of fragments using silver staining and autoradiography,
Single-stranded Conformation Polymorphism is a low-cost method for the analysis of microbial communities. SSDNA mobility not only depends on the three-dimensional structure of amplicons but also depends upon temperature and pH. DNA fragments should fall in size between 150-300 bp. SSCP is a low-cost technique based on a single-stranded rRNA gene sequence.