The reverse transcriptase

(redaction in progress)

Additives that increase reaction performance

DNA-dependent DNA polymerase activity

  • It is utilised in template switching methods to add linkers to first-strand cDNAs.
  • It is also a source of antisense artefacts when the RT makes a second-strand cDNA that is mistaken for a first-strand cDNA. ActinomycinD inhibits DNA-dependent, but not RNA-dependent polymerase activity and is used to suppress these artefacts (Perocchi et al., 2007, ?Kanamori-Katayama et al., 2011).

Terminal desoxynucleotidyl transferase (TdT) activity

Like other DNA polymerases (Clark, 1988), reverse transcriptases have a TdT activity.

  • Patel & Preston, 1994 showed that HIV RT adds one nucleotide (A > G >> T|C) on DNA/DNA duplexes, and more on DNA/RNA duplexes. Addition is favoured by increased dNTP levels. MMLV and AMV were also reported (data not shown) to add multiple nucleotides.

  • Chen & Patton, 2001 reported a TdT activity for MMLV and AMV, with a preference for adding As. For MMLV, activity reduced abruptly between 45 and 50 °C. For AMV, it decreased constantly from 25 to 50 °C. Activity increased with concentration for MMLV. (High-concentration AMV was not available.)

  • Oz-Gleenberg et al, 2011 showed that the the reverse transcriptase of the long terminal repeat retrotransposon Tf1, like other DNA polymerases, also adds non-templated As to blunt DNA duplexes.

Templated TdT activity

Surprisingly, reverse transcriptases can also extend cDNAs using a single nucleotide as a template.

  • Following an initial observation of Clark et al (1987) on the Klenow fragment, Ohtsubo et al, 2017a showed that specific tailing is enhanced by the complementary dNMP (C enhanced by dGMP, etc.), except for A-tailing, which is already the strongest.

  • Other nucleotides than dNMPs can enhance tailing. In particular, rA, dA, dG and dC potently induce tailing, and GMP, GDP and CDP induce continuous tailing when longer reaction times are allowed (Ohtsubo et al., 2017b).

The 5′ cap enhances C-tailing

The 5′ cap is reverse-transcribed

  • Hirzmann et al., 1993 observed the presence of an extra G at the 5′ end of cDNA clones, and concluded that the cap can be reverse-transcribed. They supported their conclusion with molecular modelling.

  • ?Volloch et al, 1995 studied cap transcription (but I could not access the article).

  • Ohtake et al, 2004 synthethised RNAs with A-caps and showed that they are reverse-transcribed as Ts.

  • Lavie et al, 2004 found extra Gs at the ends of genomic sequences of retrotransposons, showing that endogenous reverse-transcriptases also reverse-transcribe the cap.

  • Zhang et al, 2017 published a structure of an RNA-GpppG complex that suggests that a m7GpppNm / DNA duplex could form during the reverse-transcription of the cap

Reverse-transcriptases tolerate terminal mismatches

Reverse-transcription primers

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples.

BMC Genomics. 2010 Jul 2;11:413. doi:10.1186/1471-2164-11-413

Kapteyn J, He R, McDowell ET, Gang DR.

Incorporation of non-natural nucleotides into template-switching oligonucleotides reduces background and improves cDNA synthesis from very small RNA samples.

TSO starting with iCiGiC (iso-dC / iso-dG) does not form concatenates because the RTase is inhibited by these nucleotides, therefore it does not reach the end and no extra template switching occurs.

Compounds that enhance the tailing activity of Moloney murine leukemia virus reverse transcriptase.

Sci Rep. 2017 Jul 26;7(1):6520. doi:10.1038/s41598-017-04765-8

Ohtsubo Y, Nagata Y, Tsuda M.

Compounds that enhance the tailing activity of Moloney murine leukemia virus reverse transcriptase.

rA, r/dG, r/dC are potent enhancers of tailing. In longer reactions, GMP, GDP or CDP promote continuous extension of the tail. Reactions performed with 100 fmols of substrate DNA, 50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl2, 2 mM DTT, 4 mM dATP, dCTP, dGTP, or dTTP, 4 mM MnCl2, and 50 U MMLV-RT, at 30 °C.

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Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase.

Sci Rep. 2017 Feb 2;7:41769. doi:10.1038/srep41769

Ohtsubo Y, Nagata Y, Tsuda M.

Efficient N-tailing of blunt DNA ends by Moloney murine leukemia virus reverse transcriptase.

Specific tailing of first-strand cDNAs is robustly enhanced by the complementary dNMP (C enhanced by dGMP, etc.). A-tailing is not enhanced, perhaps because it is already very strong. Reactions made in presence of manganese; not tested with only magnesium.

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Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends.

Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):549-53 doi:10.1073/pnas.91.2.549

Patel PH & Preston BD.

Marked infidelity of human immunodeficiency virus type 1 reverse transcriptase at RNA and DNA template ends.

On DNA/DNA blunt ends, adds a single nucleotide (A > G >> C|T). On RNA/DNA blund ends, adds more nucleotides. Addition is favoured by increased dNTP levels.

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Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5'-RACE and primer extension.

Biotechniques. 2001 Mar;30(3):574-80, 582

Chen D, Patton JT.

Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5'-RACE and primer extension.

Terminal desoxynucleotidyl transferase activity on double-stranded substrates of reverse transcriptases MMLV and AMV: preference for adding As. For MMLV, activity reduced abruptly between 45 and 50 °C. For AMV, it decreased constantly from 25 to 50 °C. Activity increased with concentration for MMLV. (High-concentration AMV was not available.)

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Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.

Lee YH, Hsueh YW, Peng YH, Chang KC, Tsai KJ, Sun HS, Su IJ, Chiang PM.

BMC Biol. 2017 Mar 21;15(1):22. doi:10.1186/s12915-017-0359-5

Low-cell-number, single-tube amplification (STA) of total RNA revealed transcriptome changes from pluripotency to endothelium.

Poly-A tailing followed by template switching. Increased dNTPs to 2 mM and Mg2+ to 9 mM to favour terminal addition of nucleotides. TSO and RT primers are at 1 μM final.

Quantitative analysis of mRNA amplification by in vitro transcription.

Nucleic Acids Res. 2001 Mar 1;29(5):E29

Baugh LR, Hill AA, Brown EL, Hunter CP.

Quantitative analysis of mRNA amplification by in vitro transcription.

T7 pol generates aberrant template-unrelated products. This is fixed by reducing the concentration of oligo dT primers, and of enzymes. Under these conditions, 2 rounds of RNA linear amplification is not generatng too much bias. Small-volume protocol. T4gp32, a single stranded protein, increases RT processivity.

Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase.

Nucleic Acids Res. 2017 Sep 6;45(15):9046-9058. doi:10.1093/nar/gkx633

Agudo R, Calvo PA, Martínez-Jiménez MI, Blanco L.

Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase.

“PrimPol as a DNA primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer primers.”

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Kv4.2 mRNA abundance and A-type K(+) current amplitude are linearly related in basal ganglia and basal forebrain neurons.

J Neurosci. 2000 Jan 15;20(2):579-88.

Tkatch T, Baranauskas G, Surmeier DJ.

Kv4.2 mRNA abundance and A-type K(+) current amplitude are linearly related in basal ganglia and basal forebrain neurons.

Accute dissociation of slices before cell isolation. Variablility between RT lots? Evaluation of mRNA abundance through a serial dillution and callibration approach.

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Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR.

Zhang J1, Byrne CD.

Biochem J. 1999 Jan 15;337 ( Pt 2):231-41. doi:10.1042/bj3370231

Differential priming of RNA templates during cDNA synthesis markedly affects both accuracy and reproducibility of quantitative competitive reverse-transcriptase PCR.

cDNA yield did not increase with higher concentrations of specific hexamers. Higher yields with random hexamers. No data for reverse-transcription without primers.

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Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions.

Lekanne Deprez RH, Fijnvandraat AC, Ruijter JM, Moorman AF.

Anal Biochem. 2002 Aug 1;307(1):63-9.

Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions.

DTT interferes with fluorescence measurements in qPCR. RT without DTT is efficient. Random-primerd replicates are less reproducible than oligo-dT or gene-specific-primed ones.

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Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

Oz-Gleenberg I, Herzig E, Hizi A.

FEBS J. 2012 Jan;279(1):142-53. doi: 10.1111/j.1742-4658.2011.08406.x

Template-independent DNA synthesis activity associated with the reverse transcriptase of the long terminal repeat retrotransposon Tf1.

Reverse-transcriptases, add non-templated As to blunt DNA duplexes.

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Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

Bengtsson M, Hemberg M, Rorsman P, Ståhlberg A.

BMC Mol Biol. 2008 Jul 17;9:63.

Quantification of mRNA in single cells and modelling of RT-qPCR induced noise.

PCR noise is much smaller than biological noise for transcripts more abundant than 100 mRNA or 20 cDNA copies. Reverse-transcription is possible in 40 mM guanidine thiocyanate. Reported RT efficiencies vary between 99 and 2 %

Comparison of reverse transcriptases in gene expression analysis.

Clin Chem. 2004 Sep;50(9):1678-80.

Ståhlberg A, Kubista M, Pfaffl M.

Comparison of reverse transcriptases in gene expression analysis.

Estimate yield by using reference DNA and RNA molecules of the same sequence. Best: SuperScript III; worst: AMV. cDNAs were reverse-transcribed at the temperature recommended by the manufacturer, so this is probably a confounding factor.

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Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine.

Küpfer PA, Leumann CJ.

Nucleic Acids Res. 2011 Nov 1;39(21):9422-32.

Synthesis, base pairing properties and trans-lesion synthesis by reverse transcriptases of oligoribonucleotides containing the oxidatively damaged base 5-hydroxycytidine.

5-HOrC can be mis-reverse-transcribed as A.

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PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency.

Suslov O, Steindler DA.

Nucleic Acids Res. 2005 Nov 27;33(20):e181.

PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency.

Phenol / Chloroform / Isoamyl alcohol (PCI) treatment followed by ethanol precipitation strongly decreases Ct as well as increases the consistencies of the comparisons between different dillutions of the same samples

Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases.

Nucleic Acids Res. 1988 Oct 25;16(20):9677-86

Clark JM

Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases.

Terminal desoxynucleotidyl transferase activity of DNA polymerases, including AMV reverse transcriptase. Perference for adding As. ‘We cannot exclude the formal possibility that some of these latter +events, particularly the addition of dCMP by AMV reverse transcriptase, involve the use of coding +information made available as a result of a transient misalignment of the primer/template +substrate.’

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Nucleic Acids Res. 1999 Nov 1;27(21):e31.

Schmidt WM, Mueller MW.

CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs.

Extra cytosine are more frequently added in presence of the 5′ cap. Increasing Mg2+ to 6 mM increases the frequency of addition of more than 1 C, but only moderately. Instead, when adding BSA in the reaction, supplementing it with 1 or 2 mM Mn2+ after 1h, 3-4 extra dC residues are added to most of the first strand cDNAs. Standard reaction: 0.75 μM oligo dT (or 0.25 μM gene-specific RT primer); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; 5 mM DTT; dNTPs 1mM each; 0.1 mg BSA; 20 U RNAse inhibitor (Roche), 200 U SuperScript II; 1h at 42 °C.

The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity.

Lavie L, Maldener E, Brouha B, Meese EU, Mayer J.

Genome Res. 2004 Nov;14(11):2253-60 doi:10.1101/gr.2745804

The human L1 promoter: variable transcription initiation sites and a major impact of upstream flanking sequence on promoter activity.

The presence of an extra G in pseudogenes suggests that many reverse-transcriptases can reverse transcribe the cap.

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Digital transcriptome profiling using selective hexamer priming for cDNA synthesis.

Armour CD, Castle JC, Chen R, Babak T, Loerch P, Jackson S, Shah JK, Dey J, Rohl CA, Johnson JM, Raymond CK.

Nat Methods. 2009 Sep;6(9):647-9. doi:10.1038/nmeth.1360

Digital transcriptome profiling using selective hexamer priming for cDNA synthesis.

Because of reduced complexity, many 5′ ends are difficult to cover.

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

Mizuno Y, Carninci P, Okazaki Y, Tateno M, Kawai J, Amanuma H, Muramatsu M, Hayashizaki Y.

Nucleic Acids Res. 1999 Mar 1;27(5):1345-9.

Increased specificity of reverse transcription priming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display.

Reverse transcriptase can prime when the last two bases mismatch.