Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS)

Abstract

The PCR technique often yields nonspecific products. To overcome this problem, a simple, specific and efficient method was designed: touch-up and loop incorporated primers (TULIPS)-PCR. This approach utilizes loop primers (i.e., additional nontemplate 5' sequence that self-anneals to the 3' region and inhibits initiation of polymerization). Upon heating of the reaction, the primers melt, initiating hot start. The reaction also uses touch-up pre-cycling with gradual elevation in annealing temperatures to ensure correct pairing. The method has been validated with glyceraldehyde-3-phosphate dehydrogenase (GAPD) primers, and its general applicability is demonstrated by specific amplification of the human gelatinase A transgene from genomic DNA extracted from transgenic mice tails. The TULIPS-PCR protocol is a novel method. The self-annealing primers utilized in this method offer improved specificity and more robust synthesis compared with touch-down and manual hot start PCR. It is performed without the need to open, pause or add to the reaction mixture any nonrectant components, such as wax, antibody or nonspecific dsDNA.