PCR amplification of region with many ATT repeats - (Aug/20/2007 )
I'm trying to amplify a 871 bp region with numerous ATT ( about 21) repeats and other repeats for subsequent genotyping. I have added Q solution, raised the denaturing temp to 98 but still no band appears on the gel despite trying a range of annealing temps from 51 to 62 degrees. The Tm of the primers are 60.
With a highly AT rich region such as this, you must lower the extension temperature from 68 or 72 to 64 or possibly lower. You will likely need to lengthen the extension time as well. During extension, the low AT 3' end of the polymerase fragment flops off of the template. See PMID 8628694, Su XZ, Wu Y, Sifri CD, Wellems TE, Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA. Nucleic Acids Res. 1996 Apr 15;24(8):1574-5.