Bayega A, Oikonomopoulos S, Wang YC, Ragoussis J.

Front Genet. 2022 Oct 17;13:1031355. doi:10.3389/fgene.2022.1031355.

Improved Nanopore full-length cDNA sequencing by PCR-suppression.

“We observed [a range of 2.2 to 8.3-fold increase in yield] of amplicons from the Panhandle method compared to the ONT method.” “Further, the cDNA profile of samples from Panhandle protocol showed a significantly reduced amount of molecules below 600 bp compared to ONT protocol.” “Reads generated with the ONT protocol showed a marked 3′ bias with only about 40–50% of reads showing full-length coverage of the genes”

for each sample total RNA was added together with

RNA + 1 μL of 10 μm oligo (dT) primer + 1 μL of 10 mm dNTPs + Final volume: 11.6 μL pre-RT reaction.

the reaction was incubated at 72°C 3 min. 4°C 10 min, 25°C 1 min, then held at 4°C.

A 10.4μL reverse transcription (RT) reaction containing 1 X Maxima H Buffer, 1 μL RNaseOut (NEB), 2 μL of 100 μm TSO, 2 μL of 5M Betaine (Sigma-Aldrich), and 1 μL of Maxima H reverse transcriptase was added to the pre-RT reaction and the reaction incubated as shown in Supplementary Protocol.

Following reverse transcription,

5 μL of cDNA was used in a 50 μL PCR reaction containing 1 μL of 10 μm PCR primer and 25 μL of 2x LongAmp Taq Master mix (NEB).

PCR was performed as shown in Supplementary Protocol. 20 PCR cycles were used. Following PCR, 1 μL of exonuclease (NEB) was added to each reaction and incubated for 15 min at 37°C followed by 15 min at 80°C and the samples were purified using 1x AMPure XP beads (Beckman Coulter).

Carter JG, Orueta Iturbe L, Duprey JHA, Carter IR, Southern CD, Rana M, Whalley CM, Bosworth A, Beggs AD, Hicks MR, Tucker JHR, Dafforn TR.

Proc Natl Acad Sci U S A. 2021 Aug 31;118(35):e2100347118. doi:10.1073/pnas.2100347118

Ultrarapid detection of SARS-CoV-2 RNA using a reverse transcription-free exponential amplification reaction, RTF-EXPAR.

“Binder DNA X anneals to viral RNA; the DNA strand of the DNA:RNA heteroduplex is cut by the restriction endonuclease BstNI, which acts as a nicking enzyme by cutting the DNA strand only; the released DNA strand is Trigger X, which is then amplified by EXPAR.”

Gonzalez-Pena V, Natarajan S, Xia Y, Klein D, Carter R, Pang Y, Shaner B, Annu K, Putnam D, Chen W, Connelly J, Pruett-Miller S, Chen X, Easton J, Gawad C.

Proc Natl Acad Sci U S A. 2021 Jun 15;118(24):e2024176118. doi:10.1073/pnas.2024176118

Accurate genomic variant detection in single cells with primary template-directed amplification.

“primary template-directed amplification (PTA) [...] takes advantage of the processivity, strong strand displacement activity, and low error rate of phi29 polymerase [...] exonuclease-resistant terminators are incorporated into the reaction, creating smaller double-stranded amplification products that undergo limited subsequent amplification. This transforms the reaction from exponential into a quasilinear process with more of the amplification occurring from the primary template.”

Lukyanov S.A., Lukyanov K.A., Gurskaya N.G., Bogdanova E.A., Buzdin A.A.

(2007)

Selective Suppression of Polymerase Chain Reaction and Its Most Popular Applications.

In: Buzdin A.A., Lukyanov S.A. (eds) Nucleic Acids Hybridization Modern Applications. Springer, Dordrecht. https://doi.org/10.1007/978-1-4020-6040-3_2

Mentions that the “SSP [selective suppression PCR] effect does not inhibit, or inhibits sparingly, amplification of very long DNA ([...] usually 6–8 kbp).”

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).

Eberwine J, Yeh H, Miyashiro K, Cao Y, Nair S, Finnell R, Zettel M, Coleman P.

Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):3010-4. doi:10.1073/pnas.89.7.3010

Analysis of gene expression in single live neurons.

Adds a second round of amplification.

Kawai Y, Kimura Y, Lezhava A, Kanamori H, Usui K, Hanami T, Soma T, Morlighem JÉ, Saga S, Ishizu Y, Aoki S, Endo R, Oguchi-Katayama A, Kogo Y, Mitani Y, Ishidao T, Kawakami C, Kurata H, Furuya Y, Saito T, Okazaki N, Chikahira M, Hayashi E, Tsuruoka S, Toguchi T, Saito Y, Ban T, Izumi S, Uryu H, Kudo K, Sakai-Tagawa Y, Kawaoka Y, Hirai A, Hayashizaki Y, Ishikawa T.

PLoS One. 2012;7(1):e30236. doi: 10.1371/journal.pone.0030236 doi: 10.1371/journal.pone.0030236

One-step detection of the 2009 pandemic influenza A(H1N1) virus by the RT-SmartAmp assay and its clinical validation.

Inactivation in 5% SDS, gel filtration on Sephacryl S-400 HR in Micro-Bio-Spin colum, and RT-SmartAmp AMV RTase, Aac DNA pol.

Nucleic Acids Res. 2018 Jan 25;46(2):538-545. doi:10.1093/nar/gkx1238

Joffroy B, Uca YO, Prešern D, Doye JPK, Schmidt TL.

Rolling circle amplification shows a sinusoidal template length-dependent amplification bias.

Small circles of length ~70-75, 80-85, ... are very hard to amplify with Phi29, and to a lesser extent with Bst 2.0

Picher ÁJ, Budeus B, Wafzig O, Krüger C, García-Gómez S, Martínez-Jiménez MI, Díaz-Talavera A, Weber D, Blanco L, Schneider A.

Nat Commun. 2016 Nov 29;7:13296. doi:10.1038/ncomms13296

TruePrime is a novel method for whole-genome amplification from single cells based on TthPrimPol.

Isothermal amplification for 3 h at 30 °C using a mixture of TthPrimPol for priming and and Φ29DNApol for extension. Target DNA was first denatured in an alkaline buffer.

Nucleic Acids Res. 1995 Mar 25;23(6):1087-8.

Siebert PD, Chenchik A, Kellogg DE, Lukyanov KA, Lukyanov SA.

An improved PCR method for walking in uncloned genomic DNA.

First description of suppressive PCR in English ?

Chen C, Xing D, Tan L, Li H, Zhou G, Huang L, Xie XS.

Science. 2017 Apr 14;356(6334):189-194. doi:10.1126/science.aak9787

Single-cell whole-genome analyses by Linear Amplification via Transposon Insertion (LIANTI).

Tn5 tagmentation with custom adapters ending with T7 promoters, followed by linear amplification by transcription, followed by cDNA synthesis and sequencing.

Seitz V, Schaper S, Dröge A, Lenze D, Hummel M, Hennig S.

Nucleic Acids Res. 2015 Nov 16;43(20):e135. doi:10.1093/nar/gkv694

A new method to prevent carry-over contaminations in two-step PCR NGS library preparations.

Proposes to introduce indexes during the cDNA PCR, and to partially match them during Library PCR, so that 1) the PCR is more stringent and 2) contaminations can be detected by sequence analysis.

RNA Biol. 2014;11(7):817-28. doi:10.4161/rna.29304

Turchinovich A, Surowy H, Serva A, Zapatka M, Lichter P, Burwinkel B.

Capture and Amplification by Tailing and Switching (CATS). An ultrasensitive ligation-independent method for generation of DNA libraries for deep sequencing from picogram amounts of DNA and RNA.

Pre-treatment by T4 PNK was needed when using circulating plasma RNA, probably because of phosphate or cyclo-phosphates at the 3′ end (caused by RNA cleavage). RT primer concentration scaled down according to the RNA amoounts (0.1 µM for 1 ng and 0.1 nM for 5 pg). TSO concentration: 1 µM.

Salk JJ, Sanchez JA, Pierce KE, Rice JE, Soares KC, Wangh LJ.

Anal Biochem. 2006 Jun 1;353(1):124-32 doi:10.1016/j.ab.2006.02.012

Direct amplification of single-stranded DNA for pyrosequencing using linear-after-the-exponential (LATE)-PCR.

The PPi produced during the PCR can be removed with the pyrosequencing enzymes themselves. The antarctic phosphatase from NEB was not very efficient at this task.

Inoue J, Shigemori Y, Mikawa T.

Nucleic Acids Res. 2006 May 17;34(9):e69 doi:10.1093/nar/gkl350

Improvements of rolling circle amplification (RCA) efficiency and accuracy using Thermus thermophilus SSB mutant protein.

By weakening the DNA-protein interaction, the authors acheived the suppression of the non-temlated products in rolling circle amplification.

Mol Biotechnol. 2002 Nov;22(3):223-30 doi:10.1385/MB:22:3:223

Reddy MK, Nair S, Sopory SK.

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis.

Ligating a DNA adaptor 3′ to the FScDNA using T4 RNA ligase.

Biotechniques. 2003 Feb;34(2):386-8, 390, 392-3

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

Probe generation directly from small numbers of cells for DNA microarray studies.

Uses T3N9 random nonamer amplification rounds (up to 5), after an initial T7dT RT.

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.

Biotechniques. 2004 Nov;37(5):854-7

Dafforn A, Chen P, Deng G, Herrler M, Iglehart D, Koritala S, Lato S, Pillarisetty S, Purohit R, Wang M, Wang S, Kurn N.

Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis. Presentation of the Ribo-SPIA kit, which can replace T7 in vitro translation for linear amplification.

Genome Biol. 2006;7(3):R18 doi:10.1186/gb-2006-7-3-r18

Subkhankulova T, Livesey FJ.

Comparative evaluation of linear and exponential amplification techniques for expression profiling at the single-cell level.

SMART has a much lower false discovery rate (FDR), but compresses the expression ratios.

Methods. 2005 Nov;37(3):229-37 doi:10.1016/j.ymeth.2005.09.003

Ginsberg SD

RNA amplification strategies for small sample populations.

Detailed protocol for Terminal Continuation (TC).

Nucleic Acids Res. 1999 Sep 15;27(18):e23.

Shagin DA, Lukyanov KA, Vagner LL, Matz MV.

Regulation of average length of complex PCR product.

Suppression PCR amplification of a phage lambda digested by HindII on which inverted terminal repeats were ligated. Suppression effect is stronger when the PCR primer is shorter than the terminal repeats, and when the PCR primer molarity is lower (tested in a 750–0 nM range).

EMBO J. 2006 Oct 18;25(20):4933-42. doi:10.1038/sj.emboj.7601368

Imbert I, Guillemot JC, Bourhis JM, Bussetta C, Coutard B, Egloff MP, Ferron F, Gorbalenya AE, Canard B.

A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus.

This enzyme is dependant on Mn2+ and does not require a primer.

Biotechniques. 2005 Mar;38(3):451-8.

Ohara R, Kikuno RF, Kitamura H, Ohara O.

cDNA library construction from a small amount of RNA: adaptor-ligation approach for two-round cRNA amplification using T7 and SP6 RNA polymerases.

High size is preserved by ligating a 5'adapter before each amplification.

Sun Z, Chen Z, Hou X, Li S, Zhu H, Qian J, Lu D, Liu W.

PLoS One. 2008;3(11):e3701. doi:10.1371/journal.pone.0003701

Locked nucleic acid pentamers as universal PCR primers for genomic DNA amplification.

Genomic DNA amplified in a collection of fragments mimicking restriction digests using LNA pentamers and the Stoffel fragment of the Taq polymerase. Annealing at 40 °C and extension at 50 °C.

PLoS Biol. 2006 Jul;4(7):e204.

Piepenburg O, Williams CH, Stemple DL, Armes NA.

DNA detection using recombination proteins.

Uses T4 usvX recombinase and other proteins such as gp32, usvY, Bsu, Nfo, ...

Alec R. Chapman, Zi He, Sijia Lu, Jun Yong, Longzhi Tan, Fuchou Tang, X. Sunney Xie

PLoS One. 2015 Mar 30;10(3):e0120889. doi:10.1371/journal.pone.0120889

Single Cell Transcriptome Amplification with MALBAC.

0.45 % IGEPAL CA-630. T4 GP32

Jaitin DA, Kenigsberg E, Keren-Shaul H, Elefant N, Paul F, Zaretsky I, Mildner A, Cohen N, Jung S, Tanay A, Amit I.

Science. 2014 Feb 14;343(6172):776-9. doi: 10.1126/science.1247651.

Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types.

Amplification by in vitro transcription. Many cells were shallow-sequenced.

Huang H, Goto M, Tsunoda H, Sun L, Taniguchi K, Matsunaga H, Kambara H.

Nucleic Acids Res. 2013 Oct 18.

Non-biased and efficient global amplification of a single-cell cDNA library.

Detection limit: 5–10 copies per cell

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

Nat Biotechnol. 2002 Jul;20(7):738-42.

Amine-modified random primers to label probes for DNA microarrays.

Incorporates aminoallyl-dUTP instead of cy3/5dUTP. Uses priming hexamers with a amino C6dT in 5′. This allows to reduce by 10 folds the quantity of RNA as a starting material. Presence of amplification products of t/rRNAs is not a problem when « optimal » quantities of total RNA are used.

Gu H, Breaker RR.

Biotechniques. 2013 Jun;54(6):337-43. doi: 10.2144/000114009

Production of single-stranded DNAs by self-cleavage of rolling-circle amplification products.

Produces a ladder.

Biotechniques. 2011 Mar;50(3):177-80.

Method for improved Illumina sequencing library preparation using NuGEN Ovation RNA-Seq System.

Head SR, Komori HK, Hart GT, Shimashita J, Schaffer L, Salomon DR, Ordoukhanian PT.

Digests possible terminal hairpins with nuclease S1, in order to increase yield of blunting and end repair step.

Che S, Ginsberg SD.

Lab Invest. 2004 Jan;84(1):131-7.

Amplification of RNA transcripts using terminal continuation.

Templates switching with a DNA oligonucleotide ending in a mixture of Gs and Cs.

Nat Biotechnol. 2000 Apr;18(4):457-9 doi:10.1038/74546

Wang E, Miller LD, Ohnmacht GA, Liu ET, Marincola FM.

High-fidelity mRNA amplification for gene profiling.

Combines template switch with aRNA amplification

Removes the primer artefacts by physical separation on beads.

The T4gp32 protein is directly added to the RNA.

Neurochem Res. 2002 Oct;27(10):981-92

Ginsberg SD, Che S.

RNA amplification in brain tissues.

Comparison of template swithching oligos ending in -AAA, -CCC, -GGG or -TTT. No qualitative difference for the CCC and GGG variants were reported.

Size fractionnation using 2D agarose gel.

Slow ramp time from melting to annealing normalises concentrations towards 1:1 ratio at high cycles.

RNA is reverse-transcribed with oligo dT primers carrying a viral RNA pol. promoter, the first strand cDNAs are A-tailed, the second-strand cDNAs are synthethised with oligo dT primers carrying another viral promoter, and after PCR amplification probes are synthethised by in vitro transcription.

Primary paper for aRNA.

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.

High concentration of a dT and random primers mix in reverse transcription.

Dai ZM, Zhu XJ, Chen Q, Yang WJ.

J Biotechnol. 2007 Feb 20;128(3):435-43. doi:10.1016/j.jbiotec.2006.10.018

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

PCR amplification of a GeneRuler 1kb DNA Ladder (Fermentas) on which adatpers were ligated. Investigates “parameters which affect ITR self-annealing: (i) the length of PCR products [...] (ii) Primer concentration [...] (iii) The ratio of ITR length to primer length [and] (iv) [annealing temperature]”. Uses a 3′ phosphate to prevent the template switching oligonucleotide to prime a first strand cDNA.

“The 10 µl final [RT] reaction mixture contained 50 mM Tris–Cl (pH 8.3 at 25°C), 75 mM KCl,6 mM MgCl2, 2 mM MnCl2, 0.2 mg/ml BSA, 10 mM DTT, 1 mM dNTPs, 1µM TS-oligo, 1µM oligo(dT) adaptor (, 10 U RNase Inhibitor, and 150 U SuperScript II [and] was incubated at 42 °C for 1 h, followed by 45 °C for 30 min, and 50 °C for 10 min.”

“Using [...] the shortest adaptor, whose length is equal to [the PCR primer], short fragments corresponding to 0.25 kb were efficiently amplified, while longer fragments (>3.5 kb) could not be distinguished from the background. Using [...] the longest adaptor, whose length is more than twice [the PCR primer], fragments shorter than 1.5 kb were suppressed, while fragments up to 10 kb were efficiently amplified.” [My comment: this analysis does not take into account the overall reduction of PCR efficiency with increasing and the fact that mass of shortest fragments were also lower in the GeneRuler ladder. This also contributes to the disappearance of these bands on the electrophoresis pictures. This might explain why the high-length fragments could not resolve in the amplifications with highest yields.]

“Lower [annealing temperature] enhanced the PS-effect under any condition. [Annealing temperature] greatly affected the PS-effect when the primer concentration was low (P2 = 0.04M). [The] average length of the products was longer at lower [annealing temperature]. [...] From 50 to 61.2°C), longer products (up to 10 kb) wer eefficiently amplified. When [annealing temperature] was higher (>65.9°C), much shorter products (<4 kb) were efficiently amplified and longer products disappeared. [Annealing temperature] also affected the PS-effect when primer concentration was 15-fold higher [...] The average product length between higher (70◦C) and lower (60◦C) [annealing temperature] was significantly different, regardless of adaptors used.”

“[Only] with the longest adaptor [...] we were able to amplify products in a wide range (from 0.25 to 10 kb) simultaneously with a slight over-representation of longer molecules. [To] representatively amplify a complex sample, relatively long ITR (compared to primer length), high [annealing temperature], and high concentratio nof primer should be used.”

Addition of unrelated, but distinguishible RNA to a sample can raise the overall concentration and stimulate the reaction.

Capable to use dUTP and to a lower extent, dITP.

“Ethylene glycol and 1,2-propanediol were found to be more effective than betaine.”

Suppressive PCR.

See also patent US5565340.