Preparing of single-stranded DNA in single-stage PCR with low-melt excess primer for hybridization on biochips

Abstract

A method for fluorescently labeled single-stranded DNA (ssDNA) production during single-stage polymerase chain reaction (PCR) for subsequent hybridization on a biochip was described. This approach, whose efficiency was confirmed in the case of DARC gene, is considered as an alternative to two-stage nested PCR, consisting of two separate reactions: symmetric and asymmetric. Implementation of PCR in a single stage was achieved due to the use of a truncated excess primer in the second stage that does not anneal on the matrix during the cycles of symmetric stage of PCR and that enters the reaction after decrease of the annealing temperature in asymmetric stage. As a result, high efficiency of genotyping by means of hybridization on biochips is maintained. The suggested approach will allow us to reduce the time, working hours, and risk of contamination when researching biochips.