Fast PCR for High-GC Amplification
As with multiplex PCR, templates with high GC content or secondary structure are often challenging to amplify. To compare the TAQXpedite System with three other suppliers’ fast PCR kits, a 270 bp sequence that is 80% GC-rich was amplified from the human FMR1 (Fragile X) gene.
PCR amplifications were set up according to the manufacturer’s directions, including the reactivation of hot-start enzymes where applicable. Each reaction included 1X MasterMix, 12.5 pmol each of forward and reverse primers, and 50 ng of human genomic DNA. The cycling conditions were 98°C for 30 seconds, followed by 35 cycles: 98°C for 5 seconds and 68°C for 20 seconds.
Only the reaction containing TAQXpedite’s Difficult/Long MasterMix resulted in the desired 270 bp amplicon in 44 minutes. The other fast PCR systems yielded nonspecific products and a background smear (Figure 4).
Use of the TAQXpedite PCR System reduced PCR cycling times when compared to traditional cycling protocols. We have shown that routine PCR of a 500-bp fragment was completed in just 16 minutes. Improved cycling times were also achieved for a variety of challenging applications including amplification of templates 30 kb or greater in length, multiplex PCR, and amplification of GC-rich templates. Neither specificity nor sensitivity were compromised when the fast PCR format was used.