Determination of Allele Frequency in Pooled DNA: Comparison of Three PCR-Based Methods

Abstract

Determination of allele frequency in pooled DNA samples is a powerful and efficient tool for large-scale association studies. In this study, we tested and compared three PCR-based methods for accuracy, reproducibility, cost, and convenience. The methods compared were: (i) real-time PCR with allele-specific primers, (ii) real-time PCR with allele-specific TaqMan probes, and (iii) quantitative sequencing. Allele frequencies of three single nucleotide polymorphisms in three different genes were estimated from pooled DNA. The pools were made of genomic DNA samples from 96 cases with basal cell carcinoma of the skin and 96 healthy controls with known genotypes. In this study, the allele frequency estimation made by real-time PCR with allele-specific primers had the smallest median deviation (MD) from the real allele frequency with 1.12% (absolute percentage points) and was also the cheapest method. However; this method required the most time for optimization and showed the highest variation between replicates (SD = 6.47%). Quantitative sequencing, the simplest method, was found to have intermediate accuracies (MD = 1.44%, SD = 4.2%). Real-time PCR with TaqMan probes, a convenient but very expensive method, had an MD of 1.47% and the lowest variation between replicates (SD = 3.18%).

Liquid biopsy in early stage lung cancer

“…Nowadays, there are various methods to detect genetic alterations in cfDNA, including real-time polymerase chain reaction (PCR), digital PCR, amplification protocols with magnetic beads in oil emulsions (beads emulsion), amplification and magnetics (BEAMing), next-generation sequencing (NGS) and mass spectrometry (MS) genotyping (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34). The limit of mutation detection of these techniques fluctuates from 15% to 0.01%, but one of the main problems is the absence of standardized methods for biospecimen collection, processing, cfDNA isolation and analysis.…”

“…Three types of real-time PCR are routinely employed, based on the probes utilized: Taqman probes, Taqman Detection Mutation Assay and Scorpion probes. Taqman probes have a mutation detection limit of around 10%, whereas Taqman Detection Mutation Assay may detect mutations as low as 0.1% (23,24). These variations in sensitivity are due to the probe design: Taqman probes consist of a fluorophore covalently attached to the 5'-end of the oligonucleotide probe and a quencher at the 3'-end, whereas Taqman Detection Mutation Assay uses an allele-specific primer and an MGB blocker oligonucleotide that suppresses the wildtype background (35).…”

Relative quantitation of virus population size in mixed genotype infections using sequencing chromatograms

“…After confirming that the peaks correspond to the known variation in the SNP, the peak areas and heights were extracted and the relative peak areas or heights were calculated for each virus species. The relative proportions (PAV / (PAV + PAS)) were used instead of the ratio of the relative peak heights (PAV/PAS), as has been done in previous studies (Wilkening et al, 2005), because this study was interested in relative viral transcript concentration rather than the relative magnitude of separation between concentrations.…”

“…As such, they can only determine if the end result of competition is coexistence or complete exclusion. Measurement of the relative area or height of sequencing chromatogram peaks at polymorphic sites can result in quantitative data if a standard curve is constructed by mixing known amounts of viral template and performing PCR amplification followed by direct sequencing (Wilkening et al, 2005). Direct nucleotide sequencing has two major advantages over the previously listed methods.…”

“…Quantitative sequencing has previously been used to estimate allele frequency and mutation frequency in pooled DNA samples (Amos et al, 2000, Wilkening et al, 2005. The data are quantitative because the height or area of the peaks in a sequencing chromatogram at polymorphic nucleotide positions are proportional to the corresponding template concentration at the start of the sequencing reaction.…”