Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing

Abstract

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.