Linear-After-The-Exponential (LATE)–PCR: An advanced method of asymmetric PCR and its uses in quantitative real-time analysis

Abstract

Conventional asymmetric PCR is inefficient and difficult to optimize because limiting the concentration of one primer lowers its melting temperature below the reaction annealing temperature. Linear-After-The-Exponential (LATE)–PCR describes a new paradigm for primer design that renders assays as efficient as symmetric PCR assays, regardless of primer ratio. LATE-PCR generates single-stranded products with predictable kinetics for many cycles beyond the exponential phase. LATE-PCR also introduces new probe design criteria that uncouple hybridization probe detection from primer annealing and extension, increase probe reliability, improve allele discrimination, and increase signal strength by 80–250% relative to symmetric PCR. These improvements in PCR are particularly useful for real-time quantitative analysis of target numbers in small samples. LATE-PCR is adaptable to high throughput applications in fields such as clinical diagnostics, biodefense, forensics, and DNA sequencing. We showcase LATE-PCR via amplification of the cystic fibrosis CF Δ 508 allele and the Tay-Sachs disease TSD 1278 allele from single heterozygous cells.

Closed-Tube Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye

“…The robustness of asymmetric PCR has recently been questioned (25 ). The decreased concentration of the limiting primer lowers its T m compared with the excess primer, a fact that may be overlooked.…”

A Homogeneous HLA-B*27 Genotyping Assay Using Dried Reagent Mixtures

“…The primers amplify all HLA-B*27-alleles shown to be associated with AS; only few rare alleles are not amplified. A minor modification was done to the HLA-B*27-specific 5'-primer by adding four bases to the 5'-end of the primer to compensate for the diminished concentration of this primer used in the asymmetric PCR when compared to the HLA-B*27-specific 3'-primer concentration, since the T m of the primers depends on the concentration as well as the length of the primer [8,15]. The inclusion of LNA-bases in the lanthanide-labeled probes was shown by our previous work to improve significantly the performance of the probes, when compared to regular, only-DNA containing oligonucleotides, in the developed assay format [8] and although the specificity in this assay relies mainly on the HLA-B*27-primers, given that the HLA-B*27 probe used in the homogeneous assay has a sequence which can be found also in other HLA-B-alleles, such as HLA-B*07, *08 and *39 among others, we chose to take advantage of the strong affinity of LNA-bases [2,12] in this assay as well to allow the use of short probes.…”

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)–PCR

“…LATE-PCR primer sets were designed to amplify a 191 base-pair region containing the β-globin IVS 110 site (human chromosome 11p), a 95 base pair region containing the rs858521 SNP site (human chromosome 17p) and a 78 base pair region containing the rs2270517 SNP site (human chromosome 17p) using previously described methods [29,30,32]. Briefly, primer pairs were selected such that ?…”

“…Liner-After-The-Exponential (LATE]-PCR is a novel, highly robust asymmetric PCR method that uses unequal concentrations of primers to generate large amounts of single-stranded DNA (ssDNA) together with a small fixed amount of double-stranded DNA (dsDNA) [29,30]. Although the concept of asymmetric PCR has been in the literature for more than fifteen years [31], optimizing reactions to be efficient and reproducible has historically been problematic.…”