The reverse transcriptase
(redaction in progress)
Additives that increase reaction performance
- Actinomycin D (Perocchi et al., 2007).
- T4 bacteriophage gene 32 protein (T4gp32, Kenzelmann et al. , 2004, Piché et al. , 2005).
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, ).
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
- Schmidt & Mueller, 1999 showed that extra cytosine are more frequently added in presence of the 5′ cap.
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.
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
- Reported by Mizuno et al., 1999.
- Utilised in Arnaud et al., 2016 to reduce priming or ribosomal or hemoglobin RNA.
- "N15" random pentadecamers: Stangegaard et al., 2006.
- Multi-targeted primers (MTP): Adomas et al., 2010.
- "Not-so random" (NSR) primers: Armour et al., 2009.
- "Pseudo-random" primers: Arnaud et al., 2016.
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.
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.
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.
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.
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.)
Arnaud O, Kato S, Poulain S, Plessy C.
Biotechniques. 2016 Apr 1;60(4):169-74. doi:10.2144/000114400
Targeted reduction of highly abundant transcripts using pseudo-random primers.
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.
Chen Y, Zhong JF.
Methods Mol Biol. 2008;438:293-303. doi:10.1007/978-1-59745-133-8_22
Microfluidic devices for high-throughput gene expression profiling of single hESC-derived neural stem cells.
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.
J Biomol Tech. 2005 Sep;16(3):239-47
Piché C, Schernthaner JP.
Optimization of in vitro transcription and full-length cDNA synthesis using the T4 bacteriophage gene 32 protein.
Nucleic Acids Res. 2007;35(19):e128 doi:10.1093/nar/gkm683
Perocchi F, Xu Z, Clauder-Münster S, Steinmetz LM.
Antisense artifacts in transcriptome microarray experiments are resolved by actinomycin D.
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.
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.
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.
Nat Methods. 2015 Sep;12(9):835-7. doi:10.1038/nmeth.3478
Zheng G, Qin Y, Clark WC, Dai Q, Yi C, He C, Lambowitz AM, Pan T.
Efficient and quantitative high-throughput tRNA sequencing.
Cozen AE, Quartley E, Holmes AD, Hrabeta-Robinson E, Phizicky EM, Lowe TM.
Nat Methods. 2015 Sep;12(9):879-84. doi:10.1038/nmeth.3508
ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments.
Ruprecht RM, Goodman NC, Spiegelman S.
Biochim Biophys Acta. 1973 Jan 19;294(2):192-203.
Conditions for the selective synthesis of DNA complementary to template RNA.
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.
Taggart AJ, DeSimone AM, Shih JS, Filloux ME, Fairbrother WG.
Nat Struct Mol Biol. 2012 Jun 17;19(7):719-21. doi: 10.1038/nsmb.2327
Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo.
Zajac P, Islam S, Hochgerner H, Lönnerberg P, Linnarsson S.
PLoS One. 2013 Dec 31;8(12):e85270. doi: 10.1371/journal.pone.0085270
Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases.
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.
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 %
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.
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.
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
Nucleic Acids Res. 1988 Oct 25;16(20):9677-86
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.’
J Biosci Bioeng. 2005 Mar;99(3):293-5. doi:10.1263/jbb.99.293
Nakano M, Nakai N, Kurita H, Komatsu J, Takashima K, Katsura S, Mizuno A.
Single-molecule reverse transcription polymerase chain reaction using water-in-oil emulsion.
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.
Kenzelmann M, Klären R, Hergenhahn M, Bonrouhi M, Gröne HJ, Schmid W, Schütz G.
Genomics. 2004 Apr;83(4):550-8 doi:10.1016/j.ygeno.2003.09.026
High-accuracy amplification of nanogram total RNA amounts for gene profiling.
DNA Res. 2004 Aug 31;11(4):305-9
Ohtake H, Ohtoko K, Ishimaru Y, Kato S.
Determination of the capped site sequence of mRNA based on the detection of cap-dependent nucleotide addition using an anchor ligation method.
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.
Biotechniques. 2006 May;40(5):649-57.
Stangegaard M, Dufva IH, Dufva M.
Reverse transcription using random pentadecamer primers increases yield and quality of resulting cDNA.
Nucleic Acids Res. 2006;34(17):4702-10. doi:10.1093/nar/gkl625
Leal NA, Sukeda M, Benner SA.
Dynamic assembly of primers on nucleic acid templates.
Nucleic Acids Res. 2006;34(21):e143 doi:10.1093/nar/gkl740
Hartmann CH, Klein CA.
Gene expression profiling of single cells on large-scale oligonucleotide arrays.
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.
Nucleic Acids Res. 1993 Jul 25;21(15):3597-8
Hirzmann J, Luo D, Hahnen J, Hobom G.
Determination of messenger RNA 5'-ends by reverse transcription of the cap structure.
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.