Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini.

Creighton, Steven; Huang, Meimei; Cai, Hong; Arnheim, Norman; Goodman, Myron F.

doi:10.1016/s0021-9258(18)45928-5

Cited by 78 publications

(14 citation statements)

References 31 publications

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“…That presumption is partially supported by the evidence that A•C mispair formation with a DNA primer was ∼10 times lower on a RNA compared with a DNA template of the same sequence (7). It was determined that the extension efficiency for the mismatched primer terminus was >5 orders of magnitude lower than that of the matched one (40). Therefore, formation and extension of a mismatched primer terminus is an inefficient process on DNA-primed RNA.…”

mentioning

confidence: 96%

Differences in Mutagenesis During Minus Strand, Plus Strand and Strand Transfer (Recombination) Synthesis of the Hiv-1 Nef Gene in Vitro

Palaniappan

Bambara

et al. 1996

Nucleic Acids Research

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We have developed an HIV nef-Escherichia coli lacZ fusion system in vitro that allows the detection of low frequency mutations, including frameshifts, deletions and insertions. A portion of the nef gene that encompasses a hypervariable region was fused in-frame with a downstream lacZalpha peptide coding region. The resulting lacZalpha peptide fusion protein remained functional. Any frameshift mutations in the nef insert would put the downstream lacZ alpha peptide gene out of frame, eliminating alpha complementation. With this system we compared the error rates of frameshift mutations that arise during DNA-directed and RNA-directed DNA synthesis. Results showed that DNA-directed and RNA-directed DNA synthesis did not contribute equally to the generation of mutations. DNA-directed DNA synthesis generated frameshift mutations at a frequency approximately 10-fold higher than those arising from RNA-directed DNA synthesis. RNA-directed DNA synthesis in the presence of acceptor templates showed an increase in mutation rate and differences in the mutation spectrum. The enhancement of mutation rate was caused by the appearance of mutations at three new locations that correlated with likely recombination sites. Results indicate that recombination is another source of mutations during viral replication.

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mentioning

confidence: 96%

Differences in Mutagenesis During Minus Strand, Plus Strand and Strand Transfer (Recombination) Synthesis of the Hiv-1 Nef Gene in Vitro

Palaniappan

Bambara

et al. 1996

Nucleic Acids Research

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“…bVf/Km is obtained by a least squares fit of the kinetic data to the linear region mismatch/correct match extension efficiency, which measures the direct competition for polymerase to extend a termimal mispair in the presence of a correct pair when both are present at equimolar concentrations, and when the polymerase binds with equal affinites to each (15). We have recently shown that KD values are similar for AMV RT binding to all natural matched and mismatched base pairs (17). One can also use equation 2 to compare rates of extending any primer-template termini.…”

Section: Resultsmentioning

confidence: 99%

A comparison of the fidelity of copying 5-methylcytosine and cytosine at a difined DNA template site

Shen¹,

Creighton²,

Jones³

et al. 1992

Nucl Acids Res

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5-Methylcytosine has been postulated to be an endogenous mutagen in procaryotes and eucaryotes leading to base substitution hot spots, C-->T transitions, resulting from spontaneous deamination of mC to T. The possibility remains, however, that a second mechanism involving mispairing of mC with A might also contribute to base substitution mutagenesis via G-->A transitions. Stimulation of the G-->A mutational pathway could involve preferential misincorporation of dAMP opposite template mC compared to C. To investigate this possibility, we synthesized a sequence containing mC at a defined template location. We compared the fidelity of copying mC versus C and the efficiency of extending mismatched base pairs at the mC position using three DNA polymerases, AMV reverse transcriptase, Drosophila DNA polymerase alpha, and mutant Escherichia coli Klenow fragment containing no proofreading exonuclease activity. Significant differences in misinsertion and mismatch extension efficiencies were observed only for the case of AMV reverse transcriptase. AMV reverse transcriptase was observed to incorporate dAMP 4 to 5-fold more efficiently opposite mC than C. Favored extension of a 5-MeC.A over C.A mispair was also observed with a difference of about 3-fold. In contrast to AMV reverse transcriptase, Klenow fragment showed no significant difference when copying either mC or C sites or when extending mispairs involving mC and C. Incorporation of dAMP opposite either C or mC was barely detectable using pol alpha, although pol alpha has been observed to form A.C mismatches in other sequences. While we cannot completely exclude the possibility that dAMP might be incorporated opposite mC in preference to C, our results suggest that contributions of the G-->A pathway to mC mutagenic hot spots are likely to be minor, lending additional support to the model invoking deamination of mC.

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“…cDNA obtained using random primers incorporates any initially mismatched nucleotides and although the efficiency of extension depends on how efficiently a primer hybridises onto its complementary target sequence [48] and how effective Taq polymerase binds to both [49], the consequences of mismatches during the PCR reaction are not straightforward [50]. A series of studies has established that the effects of mismatches are variable and depend on sequence context [49,51], the nature of the mismatch [52], reaction conditions [53,54], polymerase [55] as well as primer length [56].…”

Section: Discussionmentioning

confidence: 99%

RT-qPCR Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

Bustin

Kirvell

Huggett

et al. 2021

Preprint

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The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an established tool for the diagnosis of RNA pathogens. Its potential for automation has caused it to be used as a presence/absence diagnostic tool even when RNA quantification is not required. This technology has been pushed to the forefront of public awareness by the COVID-19 pandemic, as its global application has enabled rapid and analytically sensitive mass testing, with the first test targeting three viral genes published within days of the publication of the SARS-CoV-2 genomic sequence. One of those, targeting the RNA-dependent RNA polymerase gene, has been heavily criticised for supposed scientific flaws at the molecular and methodological level and this criticism has been extrapolated to doubts about the validity of RT-qPCR for COVID-19 testing in general. We have analysed this assay in detail and our findings reveal some limitations, but also highlight the robustness of the RT-qPCR methodology for SARS-CoV-2 detection. Whilst our data show that some errors can be tolerated, it is always prudent to confirm that primer and probe sequences complement their intended target, since when errors do occur, they may result in a reduction in the analytical sensitivity. However, in this case it is unlikely that a mismatch will result in poor specificity or significant number of false positive SARS-CoV-2 diagnoses, especially as this is routinely checked by diagnostic laboratories as part of their quality assurance.

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Base mispair extension kinetics. Binding of avian myeloblastosis reverse transcriptase to matched and mismatched base pair termini.

Cited by 78 publications

References 31 publications

Differences in Mutagenesis During Minus Strand, Plus Strand and Strand Transfer (Recombination) Synthesis of the Hiv-1 Nef Gene in Vitro

Differences in Mutagenesis During Minus Strand, Plus Strand and Strand Transfer (Recombination) Synthesis of the Hiv-1 Nef Gene in Vitro

A comparison of the fidelity of copying 5-methylcytosine and cytosine at a difined DNA template site

RT-qPCR Diagnostics: The “Drosten” Sars-Cov-2 Assay Paradigm

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