From 1c8e3ba6e05c08ab324042f5d5626e852734d4c6 Mon Sep 17 00:00:00 2001 From: Charles Date: Fri, 21 Feb 2020 13:14:04 +0900 Subject: [PATCH] =?utf8?q?Caf=C3=A9?= MIME-Version: 1.0 Content-Type: text/plain; charset=utf8 Content-Transfer-Encoding: 8bit --- biblio/7511410.mdwn | 10 ++++++++++ tags/reverse_transcription.mdwn | 8 +++++--- 2 files changed, 15 insertions(+), 3 deletions(-) create mode 100644 biblio/7511410.mdwn diff --git a/biblio/7511410.mdwn b/biblio/7511410.mdwn new file mode 100644 index 00000000..df2599b9 --- /dev/null +++ b/biblio/7511410.mdwn @@ -0,0 +1,10 @@ +[[!meta title="Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase."]] +[[!tag reverse_transcription template_switching]] + +Peliska JA, Benkovic SJ. + +Biochemistry. 1994 Apr 5;33(13):3890-5 doi:10.1021/bi00179a014 + +Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. + +[[!pmid 7511410 desc="When the extra base added by the RTase in a template-switching reaction is determined by sequencing, the result does not reflect previous results obrained by primer extension assay (where A >> C on non-capped blunt DNA/RNA ends). This may be because of the differential efficiency of the RTase to extend a template over various single-base mismatches. In this assay, T to C and G to C were more frequent than T to A and G to A. Nevertheless, the experiments support previous evidence that addition is mostly limited to a single nucleotide. RT reaction: 50 mM Tris-HCl, pH 8.0; 75 mM KCl;, 0.1 mM EDTA; 1 mM DTT; 0.1% Triton X-100; 100 µM each dNTP; 7 mM MgCl2; 200 nM 24-base DNA-40-base RNA primer-template; 700 nM 41-base RNA template 2; 700 nM 42-base RNA template 3; and 100 nM HIV-1 RT in a final volume of 10 µL. When reaction products were to be sequenced, mixtures were incubated at 37 °C for 2 h."]] diff --git a/tags/reverse_transcription.mdwn b/tags/reverse_transcription.mdwn index d57c0a65..f9dea9a2 100644 --- a/tags/reverse_transcription.mdwn +++ b/tags/reverse_transcription.mdwn @@ -36,9 +36,11 @@ _(redaction in progress)_ Like other DNA polymerases ([[Clark, 1988|biblio/2460825]]), reverse transcriptases have a TdT activity. -Peliska JA, Benkovic SJ. - -Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase. +Choice of the assay for studying TdT activity can strongly influence the results. +For instance, [[Pelisca and Benkovic, 1994|biblio/7511410]] showed strong +discrepancy between radiolabelled primer extension assay and sequencing cDNAs +that have been further extended. They suppose that differential tolerance of RT +to mismatches can be the explanation. - [[Peliska and Benkovic, 1992|biblio/1279806]] showed that HIV-1 RT adds one nucleotide on non-capped oligonucleotide duplexes. A > G > T > C ratio: -- 2.47.3