Rapid cloning of HLA-A,B cDNA by using the polymerase chain reaction: frequency and nature of errors produced in amplification.

Abstract

A method for cloning full-length HLA-A,B cDNA (1.1 kilobases) by using the polymerase chain reaction (PCR) is described. Six HLA-AB alleles (HLA-A2, -A25, -B7, -B37, -B51, and -B57) were cloned, and their structures were determined. Multiple PCR clones for each allele were sequenced to obtain both an accurate consensus sequence and an "authentic" done having that sequence. Sequences from 50 clones encoding five different alleles permit assessment of the frequency and nature of PCR-produced errors. These include recombinations, deletions, and insertions in addition to point substitutions. Authentic clones were obtained at a frequency of between 30% and 70%, and analysis of three or four clones generally should be sufficient for characterization of an allele.

Removal of polymerase-produced mutant sequences from PCR products

“…Our results are consistent with previous studies of Taq DNA polymerase fidelity, which have shown that errors producing transition mutations are much more frequent than those that yield insertion͞deletions or transversions (2,3,10,(18)(19)(20). The sequence results summarized in Table 2 indicate that the MutHLS reaction can be used to effectively remove transition mutations, and although the sample size is small, probably frameshifts and some transversions as well.…”

“…However, such errors can compromise genetic diagnostic methods based on mismatch detection with amplified DNA (8,9) and render results obtained with cloned PCR products problematic (10), with the severity of this problem increasing with DNA chain length and the number of doublings. By permitting removal of most mutant sequences generated during amplification, the method described here should prove useful in circumventing these difficulties.…”

“…However, inasmuch as the mutant frequency is a linear function of the size of the DNA segment, mutations produced by the latter polymerases become a problem when larger sequences are amplified. The significance of polymerase errors for PCR applications in genetic diagnostics and cDNA cloning has been documented in the literature (8)(9)(10).…”

Contribution of Taq polymerase-induced errors to the estimation of RNA virus diversity.

“…a change from G to A on one strand cannot be distinguished from a change from C to T occurring on the complementary strand), the twelve possible nucleotide substitutions were grouped into six categories. Distributions of Taq errors reported by Dunning et al (1988) and Ennis et al (1990) were also included for comparison. Any possible bias resulting from the nucleotide composition of the target sequence was taken into account by introducing a weighting, achieved by recalculating the number of mutations for a 50 % GjC content, before the application of the G-statistic test for heterogeneity (Sokal & Rohlf, 1995).…”

Cloning and sequencing of the cDNA for ovine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF)

“…In both techniques, it cannot be determined whether the starting cDNA represents a true genomic copy, and will depend on the number of mRNA transcripts initially present. However, errors in amplification by the PCR are infrequent events that are more likely to occur late in amplification, resulting in mutations that are unique to a single clone (19). The error rate of the PCR as calculated by Ennis et al was 1 per 1421 nucleotides sequenced.…”