SSCP Conditions Optimization

Single strand conformational polymorphism (SSCP) is a technique which is used to detect point mutation in genes. The purpose was to optimize the condition of SSCP for efficient and sensitive mutation detection in Exon 4, 5, 6, 7, 8, 9, 10, 11 and 12. All these Exon were amplified and run on agarose gel to check strong and specific amplification. These samples were then subjected for vertical electrophoresis for SSCP using different conditions of gel concentration, time, temperature and voltage to get maximum separation between single strands. Denaturation was carried out by mixing the samples with denaturant reagents. The length of nucleotide of Exon 4 was much longer than normal, so we amplified the exon 4 into two small parts named as 4a and 4b respectively after its bifurcation. The denaturation reagent consist of 50% 100mM NaOH and 50% Formamide loading buffer, 5l-10l sample was loaded in each case. In all the optimized Exons the denaturation was performed at 95C for 7 minutes in PCR machine. This optimization will prove a valuable help in further study of the Exons of the same gene and other genes for Point Mutation Detection.