Abstract
BackgroundIn conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay.ResultsTwo-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE)-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA) and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of 2:1 and 1:2 ratios of the same alleles.ConclusionSNP genotyping via Two-Temperature LATE-PCR takes place in a homogeneous closed-tube format and uses a single hybridization probe per SNP site. These assays are convenient, rely on endpoint analysis, improve the options for construction of multiplex assays, and are suitable for SNP genotyping, mutation scanning, and detection of DNA duplication or deletions.
Highlights
In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions
This paper demonstrates the utility of the Two-Temperature LATE-PCR endpoint assay by analyzing genomes that are either homozygous or heterozygous for the normal and mutant version of the G269 allele of the human hexosaminidase A (HEXA), a single G-to-A point-mutation responsible for Tay-Sachs disease (TSD)
We demonstrate the general use of Two-Temperature LATE-PCR endpoint assays by genotyping two single nucleotide polymorphisms (SNP) near the human p53 tumor suppressor gene
Summary
In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. The plateau value of symmetric PCR is unsuitable for endpoint analysis of starting target numbers because subtle differences in reaction components, thermal cycling conditions, and early mispriming events cause individual replicate samples to exit exponential amplification at slightly different times. Amplicon yield at the end of a symmetric PCR amplification varies significantly among replicates and the amount of accumulated amplicon at plateau does not reflect the amount of DNA present in the initial sample [2,3]. The coefficient of variation in amplicon yield among replicates can be as much as 45.1% [4] To overcome these limitations of endpoint analysis, realtime PCR uses a variety of detection chemistries (hybridization probes or double-stranded DNA dyes) to measure the amount of each amplicon accumulating during the exponential phase of the reaction [5,6]. Initial target numbers present in unknown samples are either measured relative to the Ct values of known target amount standards analyzed in parallel under equivalent conditions or are measured relative to themselves in sets of replicates [5,6]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
