P-10: A New Method for PCR Amplification of GC Rich Regions (Pages: 43-44)

Lachinani L *, Ghaedi K , Tanhaei S , Rabeei F , Salamian A , Yaghmaei P , Nasr Esfahani MH ,


Objective: PPARγ, a transcription factor belongs to nuclear receptor superfamily, comprises two isoforms in mouse. These two isoforms are encoded by different mRNAs which are arisen as a result of alternative promoter usage. Promoter of PPARγ1 isoform is a highly GC rich region. The promoter region of most eukaryotic genes enriched of GC nucleotides and CpG islands. During PCR amplification of these regions, GC base pairs make complex secondary structures that oppose denaturation and yield to abortion of PCR amplification. Several components like formamide, DMSO, glycerol, betaine could facilitate PCR amplification of these regions. Moreover, combinations of these materials with different PCR methods like touchdown PCR or slow down PCR are useful. Materials and Methods: Mouse genomic DNA was extracted from heart tissue by TRI reagent and dissolved in an appropriated buffer containing NaOH (8 mM). PCR reactions were performed in the presence of different amounts of AMS, DMSO, betaine and 7-deaza-dGTP. Results: By implementing a combination of AMS buffer (1X), 3% DMSO and 0.25 M betaine in PCR reactions, the PPARγ1 isoform promoter was successfully amplified. Interestingly addition of 7-deaza-dGTP, a dGTP analog, to dGTP, with 3:1 ratio removed non specific products. Conclusion: NaOH is used to decrease the formation of secondary inner structures of GC rich regions and help their denaturation during PCR amplification. Moreover, DMSO and betaine, in the absence of NaOH, could not facilitate PCR amplification efficiently. Using several materials in PCR reactions yields to non specific products while 7-deaza-dGTP removed these non specific products.