Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.

TLDR

During PCR, probe hybridization to a product strand creates a substrate for exonuclease activity. The study proposes using Thermus aquaticus DNA polymerase’s 5′‑3′ exonuclease activity to detect PCR products by generating a specific signal during amplification. A 5′‑labeled, 3′‑blocked probe hybridizes to the target sequence in PCR, and the polymerase’s exonuclease activity degrades it into distinguishable fragments during amplification. The assay demonstrates high sensitivity and specificity, outperforming more cumbersome detection methods.

Abstract

The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification. An oligonucleotide probe, nonextendable at the 3' end, labeled at the 5' end, and designed to hybridize within the target sequence, is introduced into the polymerase chain reaction assay. Annealing of probe to one of the polymerase chain reaction product strands during the course of amplification generates a substrate suitable for exonuclease activity. During amplification, the 5'----3' exonuclease activity of T. aquaticus DNA polymerase degrades the probe into smaller fragments that can be differentiated from undegraded probe. The assay is sensitive and specific and is a significant improvement over more cumbersome detection methods.