ADVANTAGES OF A NEW TAQ DNA-POLYMERASE IN MULTIPLEX PCR AND TIME-RELEASE PCR

Extensive diagnostic and scientific investigations are often restricte d by limited availability of material. Therefore, methods like multipl ex PCR strategies are needed to conserve as much sample as possible. U nfortunately, the establishment of such procedures poses several diffi culties. Here we describe the advantages of a new enzyme, AmpliTaq Gol d(TM) DNA Polymerase, in multiplex and time-release PCR. The applicati on of this thermostable recombinant Taq DNA polymerase allows the spec ific amplification of DNA/cDNA targets with very high sensitivity. Wit h our protocol, the specific amplification of 13 different cDNAs of cy tokines and cytokine receptors can be realized in three multiplex PCRs (IL-2R alpha, IL-2/15R beta, gamma(c)-chain, IL-4 and IL-4R alpha; IL -10, IL-15 and IL-15R alpha; and IL-2, IFN gamma, IL-7, IL-7R alpha an d IL-9R alpha). The novel application of AmpliTaq Gold DNA Polymerase in a time-release PCR protocol allows specific amplification of target DNA/cDNA when only limited amounts of material are available or only low-copy-number DNA/cDNA is suspected. No IL-9 cDNA can be detected in peripheral blood mononuclear cells (PBMC) in the absence of any stimu lation, thus it was difficult to amplify this target with routine PCR protocols. Here we demonstrate the reliable and reproducible amplifica tion of IL-9 cDNA in the Hodgkin's lymphoma cell line KM-H2, in PBMC a nd in stimulated PBMC. Results with AmpliTaq Gold DNA Polymerase were more sensitive and specific compared with AmpliTaq(R) DNA Polymerase, with and without manual hot-start procedure.