Improved polymerase fidelity in PCR-SSCPA

Brail, Les; Fan, Erkang; Levin, D. B.; Logan, David M.

doi:10.1016/0165-7992(93)90019-r

Cited by 15 publications

(11 citation statements)

References 8 publications

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“…We amplified the dinucleotide repeat D15S128 (AcNb Z17197) 5 in an individual previously determined to be homozygous for (CA) 18 , the allele with the highest frequency. PCR was carried out with both Taq and Pfu polymerases, followed by subcloning and sequencing of individual inserts.…”

Section: Methods and Resultsmentioning

confidence: 99%

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences

Clarke

Rebelo²,

Gonçalves³

et al. 2001

Molecular Pathology

132

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The polymerase chain reaction (PCR) is used universally for accurate exponential amplification of DNA. We describe a high error rate at mononucleotide and dinucleotide repeat sequence motifs. Subcloning of PCR products allowed sequence analysis of individual DNA molecules from the product pool and revealed that: (1) monothymidine repeats longer than 11 bp are amplified with decreasing accuracy, (2) repeats generally contract during PCR because of the loss of repeat units, (3) Taq and proofreading polymerase Pfu generate similar errors at mononucleotide and dinucleotide repeats, and (4) unlike the parent PCR product pool, individual clones containing a single repeat length produce no "shadow bands". These data demonstrate that routine PCR amplification alters mononucleotide and dinucleotide repeat lengths. Such sequences are common components of genetic markers, disease genes, and intronic splicing motifs, and the amplification errors described here can be mistaken for polymorphisms or mutations. (J Clin Pathol: Mol Pathol 2001;54:351-353)

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Section: Methods and Resultsmentioning

confidence: 99%

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences

Clarke

Rebelo²,

Gonçalves³

et al. 2001

Molecular Pathology

132

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“…Prior testing of Pfu reported an error rate of 1.6 ‫ן‬ 10 ‫6מ‬ per base per doubling using a lacI-based bacterial assay (Lundberg et al 1991), and a similar error rate of 2 ‫ן‬ 10 ‫6מ‬ was reported using a p53-based biological assay (Flaman et al 1994). However, an error rate one order of magnitude higher, ∼2.5 ‫ן‬ 10 denaturing gradient gel electrophoresis (DGGE) (Cariello and Skopek 1993) and single-strand conformation polymorphism analysis (SSCP) (Brail et al 1993). The differences in these error rates demanded clarification.…”

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confidence: 96%

“…The reaction mixture pH was 8.5 at 25°C but at 94°C the pH should be ∼6. (The pH of Tris buffers decreases at higher temperatures: ∼0.035 pH units/°C (Brail et al 1993).) Each cycle of the PCR reactions consisted of 7 sec at 94°C for DNA template denaturation, 15 sec at 50°C for template-primer hybridization, and 5 sec at 72°C for DNA polymerization.…”

Section: Pcrmentioning

confidence: 99%

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

André

Kim²,

Khrapko³

et al. 1997

Genome Res.

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The study of rare genetic changes in human tissues requires specialized techniques. Point mutations at fractions at or below 10−6 must be observed to discover even the most prominent features of the point mutational spectrum. PCR permits the increase in number of mutant copies but does so at the expense of creating many additional mutations or “PCR noise”. Thus, each DNA sequence studied must be characterized with regard to the DNA polymerase and conditions used to avoid interpreting a PCR-generated mutation as one arising in human tissue. The thermostable DNA polymerase derived fromPyrococcus furiosus designated Pfu has the highest fidelity of any DNA thermostable polymerase studied to date, and this property recommends it for analyses of tissue mutational spectra. Here, we apply constant denaturant capillary electrophoresis (CDCE) to separate and isolate the products of DNA amplification. This new strategy permitted direct enumeration and identification of point mutations created by Pfu DNA polymerase in a 96-bp low melting domain of a human mitochondrial sequence despite the very low mutant fractions generated in the PCR process. This sequence, containing part of the tRNA glycine and NADH dehydrogenase subunit 3 genes, is the target of our studies of mitochondrial mutagenesis in human cells and tissues. Incorrectly synthesized sequences were separated from the wild type as mutant/wild-type heteroduplexes by sequential enrichment on CDCE. An artificially constructed mutant was used as an internal standard to permit calculation of the mutant fraction. Our study found that the average error rate (mutations per base pair duplication) ofPfu was 6.5 × 10−7, and five of its more frequent mutations (hot spots) consisted of three transversions (GC → TA, AT → TA, and AT → CG), one transition (AT → GC), and one 1-bp deletion (in an AAAAAA sequence). To achieve an even higher sensitivity, the amount of Pfu-induced mutants must be reduced.

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“…SSCP analysis holds promise as being such a technique. In SSCP analysis, the changes in single- stranded DNA mobility are very sensitive to the physical environment within the gel, particularly the temperature and the concentration of ions and solvents (Hongyo et al, 1993;Brail et al, 1993). The minigel format used in ASAP analysis provides a broad range of temperature control for band resolution.…”

Section: Discussionmentioning

confidence: 99%

Allele-specific associated polymorphism analysis: Novel modification of SSCP for mutation detection in heterozygous alleles using the paradigm of resistance to thyroid hormone

1995

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Allele-specific polymorphism (ASAP) analysis is a modification of single-strand conformation polymorphism (SSCP) mutation screening under optimized temperature conditions in a minigel format with ethidium bromide detection. ASAP analysis was used to screen for and identify mutations within the human thyroid hormone receptor-beta (hTR-beta) gene. These mutations are the underlying cause of resistance to thyroid hormone (RTH). Eleven dissimilar known hTR-beta mutations and six previously uncharacterized mutations were accurately identified. ASAP screening extends to unique ASAP-DNA fingerprinting as an identifying signature for each novel hTR-beta mutation detected thus far. Gel-plugs from the SSCP gels containing polymorphic single-stranded DNA alleles were used without elution to prepare solid-phase sequencing templates for mutant allele PCR and sequencing (MAPS). The coupling of ASAP analysis with MAPS has eliminated many of the interpretative and technical problems associated with the sequencing of heterozygous alleles. Together, this convenient screening and sequencing methodology offers accuracy, reproducibility, speed, and the potential elimination of all radioactivity, providing a general strategy for future automated detection and characterization of genetic mutations.

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Improved polymerase fidelity in PCR-SSCPA

Cited by 15 publications

References 8 publications

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences

PCR amplification introduces errors into mononucleotide and dinucleotide repeat sequences

Fidelity and Mutational Spectrum of Pfu DNA Polymerase on a Human Mitochondrial DNA Sequence

Allele-specific associated polymorphism analysis: Novel modification of SSCP for mutation detection in heterozygous alleles using the paradigm of resistance to thyroid hormone

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