Fluorescent multiplex polymerase chain reaction analysis of four genes associated with inpaired fibrinolysis and myocardial infarction.
Multiplex polymerase chain reaction (PCR) allows for the simultaneous amplification of several genes, thereby optimizing the use of reagents and decreasing personnel time. Multiplex PCR was used to amplify four genes in one PCR reaction, demonstrating the advantage of multiplex PCR for our study since it allowed us to amplify four separate genes using only 1 microl DNA, thus maximizing 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 of many DNA fragments within one base discrimination. We have used this fluorescence methodology to analyze polymorphisms associated with either impaired fibrinolysis or myocardial infarction. These include the angiotensin converting enzyme insertion/deletion (I/D) polymorphism in intron 16 of the DCP1 gene, the Alu I/D polymorphism of the tissue plasminogen activator-25 locus in intron 8, the plasminogen activator inhibitor 4G/5G repeat polymorphism, and the variable number tandem repeat of the endothelial cell nitric oxide synthase gene, all characterized by an insertion, deletion, or repeat. The amplified products were diluted 1 : 60 and analyzed on the ABI PRISM 310 Genetic Analyzer using GeneScan software. With this method, we were able to amplify four genes using 75% less reagents and personnel time, thus demonstrating the benefit of multiplex PCR and fluorescence technology.
Renshaw
M A
MA
Hematologic Diseases Branch, Division of AIDS, STD, and TB Laboratory Research, National Centers for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
Ellingsen
D
D
Costner
B
B
Benson
J
J
Heit
J A
JA
Hooper
W C
WC
D000483:Alleles; D005342:Fibrinolysis / Q000235:genetics*; D008040:Genetic Linkage; D006801:Humans; D009203:Myocardial Infarction / Q000150:complications / Q000235:genetics*; D019001:Nitric Oxide Synthase / Q000235:genetics; D052250:Nitric Oxide Synthase Type III; D007703:Peptidyl-Dipeptidase A / Q000235:genetics; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D010959:Tissue Plasminogen Activator / Q000235:genetics
D017395:Plasminogen Activator Inhibitor 1; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D007703:Peptidyl-Dipeptidase A; D010959:Tissue Plasminogen Activator