Fluorescent multiplex polymerase chain reaction analysis of four genes associated with inpaired fibrinolysis and myocardial infarction

Mltiplex polymerase chain reaction (PCR) allows for the simultaneous amplif ication of several genes, thereby optimizing the use of reagents and decrea sing personnel time. Multiplex PCR was used to amplify four genes in one PC R reaction, demonstrating the advantage of multiplex PCR for our study sinc e it allowed us to amplify four separate genes using only 1 mul DNA, thus m aximizing the use of study DNA. As compared with conventional multiplex PCR analysis with ethidium bromide, incorporating fluorescence-labeled primers into multiplex PCR reactions facilitated accurate, simultaneous analysis o f many DNA fragments within one base discrimination. We have used this fluo rescence methodology to analyze polymorphisms associated with either impair ed fibrinolysis or myocardial infarction. These include the angiotensin con verting enzyme insertion/deletion (I/D) polymorphism in intron 16 of the DC P1 gene, the Alu I/D polymorphism of the tissue plasminogen activator-25 lo cus in intron 8, the plasminogen activator inhibitor 4G/5G repeat polymorph ism, and the variable number tandem repeat of the endothelial cell nitric o xide synthase gene, all characterized by an insertion, deletion, or repeat. The amplified products were diluted 1 :60 and analyzed on the ABI PRISMN 3 10 Genetic Analyzer using GeneScan(N) software. With this method, we were a ble to amplify four genes using 75% less reagents and personnel time, thus demonstrating the benefit of multiplex PCR and fluorescence technology. (C) 2001 Lippincott Williams & ilkins.