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

The PCR technique often yields nonspecific products. To overcome this probl em, a simple, specific and efficient method was de signed: touch-up, and lo op 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, pr e-cycling with gradual elevation in annealing temperatures to ensure correc t pairing. The method has been validated with glyceraldehyde-3-phosphate de hydrogenase (GAPD) primers, and its general applicability is demonstrated b y: specific amplification of the human gelatinase A transgene from genomic DNA extmcredfrorn 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 h ot start PCR. It is performed without the need to open, pause or add to the reaction mixture any nonreactant components, such as wax antibody or nonsp ecific dsDNA.