Chez Charles/ tags/ pages tagged reverse transcription

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).
  • Its error profile is different from other DNA polymerases (de Paz et al., 2018).

Clamping activity

  • Reverse transcriptases can bind duplexes that are only annealed through a 2-nt homology. A single nucleotide is not enough and GC-rich sequences are strongly favoured. This binding is enhanced by dGTP. The assays demonstrating this used an ELISA approach (Oz-Gleenberg, Herschhorn and Hizi, 2011).

Terminal desoxynucleotidyl transferase (TdT) activity

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

Choice of the assay for studying TdT activity can strongly influence the results. For instance, Pelisca and Benkovic, 1994 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 showed that HIV-1 RT adds one nucleotide on non-capped oligonucleotide duplexes. A > G > T > C ratio: 1 : 0.5 : 0.2 : 0.07. Addition of subsequent bases is visible but much slower.

  • 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.

  • Mohr and coll, 2013 showed that the Thermostable group II intron reverse transcriptase also adds non-templated As.

  • Bhardwaj and coll, 2020 report a reverse-transcription with very low G-addition.

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).

Reverse-transcription of the 5′ cap.

Pro:

  • 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, Schweitzer and Rits, 1995 showed that the AMV reverse-transcribes the cap, using an assay where the extension of a natural hairpin in the beta-globin cDNA can be enabled by decapping.

  • Kulpa, Topping and Telesnitsky, 1997 noted the addition of one G at the position where the 5′ end of the MMLV RNA was reverse-transcribed, with a frequency of 10 %. They also discussed if it could be that the cap was reverse-transcribed.

  • Schmidt & Mueller, 1999 showed that extra cytosine are more frequently added in presence of the 5′ cap.

  • 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.

Con:

  • Chenchick and coll., 1998 reported that non-capped oligonucleotides were extended with long (1~5) cytosine tails, and that the presene of a cap did not have a “significant influence”.

  • In Dallmeier and Neyts, 2013, where first-strand cDNAs prepared with a phosphorylated reverse-transcription primer were circularised, amplified and sequenced, there is not visible evidence for G addition.

Reverse-transcriptases tolerate terminal mismatches

  • HIV-1 RT can extend A-mismatches with various efficiencies: A:C >> A:A > A:G (Perrino and coll., 1898).
  • Reported by Mizuno et al., 1999 for double-mismatches and MMLV.
  • Utilised in Arnaud et al., 2016 to reduce priming or ribosomal or hemoglobin RNA.
  • Specificity of RT nevertheless increases with reaction temperature. For instance, Xu and coll., 2019 increased it from ~30 % at 47 °C to ~75 % at 55 °C.

Reverse-transcription primers

Reverse-transcriptase point mutations

RSS Atom
A new strategy to amplify degraded RNA from small tissue samples for microarray studies.

Xiang CC, Chen M, Ma L, Phan QN, Inman JM, Kozhich OA, Brownstein MJ.

Nucleic Acids Res. 2003 May 1;31(9):e53. doi:10.1093/nar/gng053

A new strategy to amplify degraded RNA from small tissue samples for microarray studies.

Uses T3N9 instead of T7dT during all the RT reactions. No 3' bias. Efficient on degraded RNA. Independant of polyadenylation (useful for non-polyA genes, like histones).

Posted
MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

Bhardwaj V, Semplicio G, Erdogdu NU, Manke T, Akhtar A.

Nat Commun. 2019 Jul 30;10(1):3219. doi:10.1038/s41467-019-11115-x

MAPCap allows high-resolution detection and differential expression analysis of transcription start sites.

Reports that in their reverse-transcription reaction, G-addition is very low.

RT reaction in 20 µL: 10x Buffer 2 μL; dNTP (10 mM) 1 μL → 500 µM final; MgCl2 (25 mM) 4 μL → 5 mM final; DTT (0.1 M) 2 μL → 10 mM final; RNaseOUT (40 U/μl) 1 μL; SSIII (200 U/μL) 1 μL

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Fidelity of in vitro DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

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.

When the extra base added by the RTase in a template-switching reaction is determined by sequencing, the result does not reflect previous results obtained 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.

Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

Science. 1992 Nov 13;258(5085):1112-8 doi:10.1126/science.1279806

Peliska JA, Benkovic SJ.

Mechanism of DNA strand transfer reactions catalyzed by HIV-1 reverse transcriptase.

Addition of 1 extra nucleotide happens at an apparent rate of 0.1 / min. Addition of subsequent bases is visible (Fig 3) but much slower. This addition does not requrie RNAseH activity. A > G > T > C ratio on a non-capped oligonucleotide duplex: 1 : 0.5 : 0.2 : 0.07, determined by incubating the reaction with each individual dNTP. 50 mM Tris-HCl pH 8, 75 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 200 nM DNA/RNA duplex, each dNTP at 100 µM, 7 mM MgCl2, 800 nM TSO and 200 nM HIV-1 RT, at 37 °C.

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Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

Volloch VZ, Schweitzer B, Rits S.

DNA Cell Biol. 1995 Dec;14(12):991-6 doi:10.1089/dna.1995.14.991

Transcription of the 5'-terminal cap nucleotide by RNA-dependent DNA polymerase: possible involvement in retroviral reverse transcription.

Utilises a 5′ hairpin in the beta-globin mRNA to design a cDNA extension assay where the reverse-transcribed cap creates a terminal mismatch that inhibits extension. De-capping the RNA removes the inhibition. 40 mM Tris 8.3, 40 mM KCl, 5 mM MgCl2, 2 mM DTT. AMV, 45°C

Posted
Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR.

Alex Chenchick, York Y. Shu, Luda Diatchenko, Roger Li, Jason Hill and Paul D. Siebert. (Gene Cloning and Analysis Group, CLONETECH Laboratories, Pao Alto, CA, USA).

In: Gene Cloning and Analysis by RT-PCR. Edited by Paul Siebert and James Larrick. 1998

Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR.

Reaction mixture: 1 µM RTP; 1 µM TSO; 50–1000 ng total RNA; 2 mM DTT, 1 mM dNTP, 200 U SSII in 10 µL.

DNA/RNA ends tested: HO-G, Cap-G, HO-A, Cap-A, HO-C, Cap-C, HO-T

TSOs tested: rG, rGrG, rGrGrG, rGrGrGrGrG, rUrUrU, GGG, rGrGrG in all-r oligo.

With the wild-type MMLVm the HO-G DNA/RNA duplex is tailed with 1~5 extra nucleotides (Fig 2). Using radiolabelled nucleotides suggests that they are mostly Cs. "Not shown" experiments suggest that the presence of a cap "does not significantly influence the preference of addition of these non-templated nucleotides". The consensus tail is AACCC. SSII (RNAseH-) has a lower efficiency for adding nucleotides, compared with wild-type MMLV.

Template-switching is more efficient with at least 2 rG. dG is notably less efficient and rU has no visible efficiency (Figure 2).

In 2 % of the cDNAs, RT was primed by the TSO.

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

EMBO J. 1997 Feb 17;16(4):856-65 doi:10.1093/emboj/16.4.856

Kulpa D, Topping R, Telesnitsky A.

Determination of the site of first strand transfer during Moloney murine leukemia virus reverse transcription and identification of strand transfer-associated reverse transcriptase errors.

“The results presented here support the model that +1 substitutions arise during reverse transcription via non‐templated addition followed by mismatch extension upon strand transfer. Another possibility we considered was that +1G could potentially be templated by the 7‐methyl‐G cap present on mRNAs and viral genomic RNAs (Coffin, 1996). Avian myeloblastosis virus reverse transcriptase can add a cap‐complementary C residue during cDNA synthesis on mRNA in vitro, but not when the RNA has been de‐capped (Volloch et al., 1995). However, studies with purified enzymes and model primer–templates have demonstrated that +1G can arise at template switch junctions in the absence of a 7‐methyl‐G cap (Peliska and Benkovic, 1994), and our detection of +1C mutants demonstrates that not all additions to −ssDNA could be cap‐templated.”

Posted
High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing

Nucleic Acids Res. 2018 Jul 27;46(13):e78. doi:10.1093/nar/gky296

de Paz AM, Cybulski TR, Marblestone AH, Zamft BM, Church GM, Boyden ES, Kording KP, Tyo KEJ

High-resolution mapping of DNA polymerase fidelity using nucleotide imbalances and next-generation sequencing

Magnification via Nucleotide Imbalance Fidelity (MagNIFi). Investigates Dpo4, Taq, AMV RT, Sequenase 2.0 and Phi29. Confirms previous reports that when using a DNA template, the preferred misincorporation of the AMV reverse-transcriptase is A:dCTP (but other misincorporations are also frequent).

Posted
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.

Posted
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.

Posted
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 (A >> G > C|T). Addition is favoured by increased dNTP levels.

Posted
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.)

Posted
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.

Posted
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.

Posted
Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Mohr S, Ghanem E, Smith W, Sheeter D, Qin Y, King O, Polioudakis D, Iyer VR, Hunicke-Smith S, Swamy S, Kuersten S, Lambowitz AM.

RNA. 2013 Jul;19(7):958-70. doi: 10.1261/rna.039743.113

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Also reports the addition of a non-templated A.

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. Preference 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.’

Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3'-termini for initiating DNA synthesis.

Oz-Gleenberg I, Herschhorn A, Hizi A.

Nucleic Acids Res. 2011 Feb;39(3):1042-53. doi: 10.1093/nar/gkq786

Reverse transcriptases can clamp together nucleic acids strands with two complementary bases at their 3'-termini for initiating DNA synthesis.

AT-rich cDNA tails are not triggering template-switching well. CC are the strongest. Mn₂⁺ potentiates template switching for MMLV. dGTP stabilises the clamp between the enzyme and the nucleic acids.

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.

Determination of messenger RNA 5'-ends by reverse transcription of the cap structure.

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.

The mRNA cap can template an extra C in the first strand cDNA. “50% of the cDNAs of capped mRNA molecules had an extra G”

“poly(A)+ RNA was obtained using Hybond-mAP paper (Amersham). 1 µg of poly(A)+ -RNA in 9.65 µL of water was heated to 60°C for 3 min, cooled on ice, added to 4 µL of 5 × RT buffer (1 × RT buffer is 40 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 40 mM KCl, 2 mM DTE), 3 µL dNTPs (10 mM each), 0.25 µL (10 units) of RNasin (Promega), 2.5 µL of XhoI-(dT)17 oligonucleotide primer (300 pmol, 0.5 µg/µL). and 0.6 µL (16 units) of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer). The reaction was continued at 42 °C for 2 hours, and excess XhoI-(dT)17 primer and substrates were removed by glass powder purification followed by ethanol precipitation.”

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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.