The exquisite selectivity of chemical reactions enables the study of rare DNA bases. However, chemical modification of the genome can affect downstream analysis. We report a PCR bias caused by such modification, and exemplify a solution with the synthesis and characterization of a cleavable aldehyde-reactive biotinylation probe.GRM is supported by Trinity College and Herchel Smith studentships. EAR is a Herchel Smith Fellow. The SB lab is supported by core funding from Cancer Research UK. SB is a Wellcome Trust Senior Investigator.This is the final version of the article. It first appeared from the Royal Society of Chemistry via http://dx.doi.org/10.1039/C4CC05107

Balasubramanian, Shankar

McInroy, Gordon R

Raiber, Eun-Ang

Apollo (Cambridge)

This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 12047--12049 | 12047Cite this:Chem. Commun., 2014,50, 12047Chemical biology of genomic DNA: minimizingPCR bias†Gordon R. McInroy, Eun-Ang Raiber and Shankar Balasubramanian*The exquisite selectivity of chemical reactions enables the study of rareDNA bases. However, chemical modification of the genome can affectdownstream analysis. We report a PCR bias caused by such modifica-tion, and exemplify a solution with the synthesis and characterizationof a cleavable aldehyde-reactive biotinylation probe.The application of chemical tools for the study of biologicalquestions is a key tenet of chemical biology. The importance ofsuch approaches to genomic studies has grown with the utilizationof chemically reactive small molecule probes for the mapping ofmodified bases in the genome. Such methods have exploited boththe intrinsic chemical reactivity of certain modifications, and newfunctionalities introduced by chemical treatment of nucleic acids,to install detectable and capturable tags.Methylation at the C5 position of cytosine is an establishedepigenetic mark, playing an essential role in processes such asX-chromosome inactivation, transcriptional regulation1 and trans-poson silencing.2 Its TET-mediated iterative oxidation products,5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and5-carboxycytosine (5caC), are also present in low abundance inmammalian genomes. Though less well-understood than 5-methyl-cytosine (5mC), they have now been implicated in active DNAdemethylation3 and epigenetic processes.4 Central to the growingunderstanding of these DNA base modifications has been thedevelopment of techniques for their genomic mapping, many ofwhich have involved chemical biology approaches. Enrichmentmethods for genomic mapping of the marks have been widelyused, with the majority of approaches taken employing chemicalmodification of DNA in the workflow. Such methods involveisolating fragments of the genome that contain a particular mark,followed by sequencing to reveal where in the genome it can befound. The chemical methods have exploited the particularfunctional groups of 5hmC,5–7 5fC,8,9 and 5caC,10 to install acapturable tag such as biotin. This allows for a selective enrichmentof tagged fragments with streptavidin-coated beads, before sequen-cing and bioinformatics analysis.Due to the low levels at which the modifications are present, forexample, there are approximately ten 5fC per million total cytosinespecies in a mammalian genome,11 post-enrichment PCR amplifi-cation is customarily required before DNA sequencing is possible.PCR amplification does not result in an equal distribution offragments; indeed libraries generated using PCR are known tohave a biased composition.12 Whilst this effect is often due to thesequence composition, for example GC rich sequences are knownto be underrepresented following PCR amplification, polymerasestalling at unnatural base modifications can be another source ofbias.13 Indeed it has been shown that bisulfite treated 5hmC formsan adduct, 5-methylenesulfonate, which can cause polymerasestalling and thus lead to underrepresentation of densely hydroxy-methylated regions.14 Several methods previously mentioned leavesignificant chemical scarring on DNA (Fig. 1), resulting from eitherthe entire probe being left in place or some significant chemicalresidue following cleavage. It is therefore important to considerwhether PCR amplification leads to an underrepresentation offragments containing the modification of interest in the final library.Considering the scarcity of these modifications, this may lead topeaks being lost in background noise. Herein we describe a PCR biascaused by a commercial aldehyde reactive probe, and report thesynthesis of a cleavable version that leaves minimal chemicalscarring following cleavage and resolves the bias.We synthesised a probe† (Scheme 1A) capable of installing abiotin moiety at aldehyde containing sites, as found in 5fC orgenerated from the periodate cleavage of vicinal diols such asglucose.5 Probes with such chemical reactivity have been employedin recently developed techniques for mapping 5hmC5 and 5fC.8Additionally we introduced a cleavage site at the reactive terminus toallow a near complete scission of the probe from its substrate. Forthis we utilized an azide masked hemiaminal ether, which may becleaved by Staudinger reduction, and the subsequent decompositionof the highly unstable hemiaminal ether (Scheme 1B). The moiety isDepartment of Chemistry, University of Cambridge, Lensfield Road, Cambridge,CB2 1EW, UK. E-mail: sb10031@cam.ac.uk† Electronic supplementary information (ESI) available: Synthetic procedures,molecular characterisation and oligonucleotide sequences. See DOI: 10.1039/c4cc05107fReceived 3rd July 2014,Accepted 15th August 2014DOI: 10.1039/c4cc05107fwww.rsc.org/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 18 August 2014. Downloaded on 04/09/2015 10:07:54.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View Issue12048 | Chem. Commun., 2014, 50, 12047--12049 This journal is©The Royal Society of Chemistry 2014stable to heat, pH and oxidative conditions, and yet under mild,reductive and non-denaturing conditions quantitative cleavageoccurs. This cleavage chemistry is key to Solexa/Illumina DNAsequencing, where its biocompatibility and cleavage efficiency areclearly demonstrated.15 Our probe was synthesised in five steps from2-bromomethyl-1,3-dioxolane. Initially, the cleavage site was formedby treatment with trimethylsilyl azide in the presence of a Lewis acid.The reactive hydroxylamine moiety was then installed with mesylationactivation followed by an O-alkylation of boc-protecting hydroxyl-amine to give 4. Alkaline ester hydrolysis and pentafluorophenyl(Pfp) activation allowed coupling with ethylamine modified biotin tointroduce the capture tag. A final boc-deprotection with methanolicHCl and RP-HPLC purification yielded the pure probe 1.The aldehyde reactivity and selectivity of the probe was confirmedby incubating 15-mer oligonucleotides† containing cytosine, 5mC,5hmC or 5fC with 1 in the presence of p-anisidine at pH 5. Analysisusing LC-MS revealed a DNA-probe adduct formed only between the5fC-containing DNA and 1, with the mass expected following oximeformation at the 5fC aldehyde (Fig. 2). Additionally, the efficiency ofcleavage induced by azide reduction was evaluated. We found tris-(2-carboxyethyl)phosphine (TCEP) buffered with Tris-HCl pH 7.4 to becapable of facilitating the quantitative conversion of the DNA-probeadduct to 5fC-oxime (5foxC) containing DNA within 15 min at 65 1C.To measure the effect of chemical tags on amplification effi-ciency, quantitative real-time PCR (qPCR) was employed. 100-meroligonucleotides containing one, four or ten 5fC sites were gener-ated by PCR and reacted with either a commercially availablealdehyde reactive probe16 (ARP) or probe 1. Additionally, samplesof DNA-1 adducts were further treated with TCEP to yield DNAcontaining the 5foxC cleavage product. The threshold cycle (Ct)obtained for 1 pg of DNA input was normalised against a 5fCcontrol to give a DCt value indicative of the modification inducedPCR bias, where higher values represent a decrease in amplificationefficiency. Samples treated with either ARP or 1 led to similar DCtprofiles, where low adduct numbers led to a delay of one cycle, whilehigher modification density caused a significantly greater change ofover two cycles (Fig. 3). In contrast, the strands containing the 5foxCcleavage product induced no significant DCt from the controltemplate, irrespective of the modification density, and greatlyreduced the density dependent PCR inhibition effect. These findingsshow that DNA scarring, in this case due to the presence of acapturable biotin tag, may lead to underrepresentation followinginefficient PCR amplification. This matter may be resolved by thestrategic placement of a cleavage site to minimize chemical scarring.To further investigate the apparent chemical scarring inducedPCR bias, we performed polymerase primer extension reactionswith differentially modified synthetic oligomer substrates. Thesubstrates were 100-mers with five modification sites betweenpositions fifty and sixty. Single primer extension amplificationwas performed using fluorescently labelled primers, with aliquotstaken after each cycle and run on a high resolution denaturingpolyacrylamide gel. The multi-cycle approach allowed the visualisa-tion of minor pause sites that would not be apparent after a singleprimer extension due to low intensity.Fig. 1 Structures describing examples of molecular scarring resultingfrom DNA probe interactions. Structures show increased steric bulk andunnatural functional groups. Each structure is labelled with the targetmodification and a literature reference.Scheme 1 (A) Synthesis of cleavable probe 1 (a) TMS-N3, SnCl4 (b)(i) MsCl, Et3N, DCM (ii) BocNHOH, DBU, Et2O (c) (i) NaOH, EtOH (ii) PfpTFA,Et3N, Et2O. (d) Biotin ethylenediamine, DMF (e) HCl:MeOH. (B) Reaction of1 with the aldehyde moiety and subsequent cleavage to yield the oxime.Fig. 2 LC-MS was used to follow 5fC-containing DNA (top) throughincubation with probe 1 at 37 1C for 24 h (middle), and subsequent probecleavage with TCEP at 65 1C for 15 min (bottom). Base X indicates a 5fC–1adduct; traces are base peak chromatograms.Communication ChemCommOpen Access Article. Published on 18 August 2014. Downloaded on 04/09/2015 10:07:54.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 12047--12049 | 12049The unmodified template showed only product bands (Fig. 4B-a),which confirmed there were no inherent sequence or secondarystructure induced pause sites. Likewise, the 5fC containing templatewas amplified effectively (Fig. 4B-b), though the presence of very faintbands suggests 5fC may have some effect on polymerase processivityor DNA structure. However, the small-molecule probe containing thetemplate resulted in strong polymerase pause sites (Fig. 4B-c), withtwo distinct truncation products evidently coinciding with themodified region. The pause sites suggest that the polymeraseencounters difficulty in translocating the bulky base adducts intoor through the active site.17 When probe 1 was cleaved from thetemplate to yield 5fC-oxime containing DNA, polymerase stalling wasclearly diminished (Fig. 4B-d), indicating that the near-natural basegenerated following chemical cleavage is not a major obstacle toPCR. Multiple extensions allowed visualisation of faint pause sites,suggesting that even the minimal residue could inhibit polymerasereplication to some extent. The 5fC-oxime residue adds little stericbulk, but introduces additional hydrogen bonding possibilities andalters the tautomerization equilibrium,18 which may affect the DNAstructure or polymerase processivity.In conclusion, we have shown that the presence of chemicalscarring following DNA labelling can cause DNA polymeraseinhibition. The severity of this inhibition is related to theincreasing modification density. Additionally, we have synthe-sised an aldehyde reactive biotin probe that may be removedunder mild, nucleic acid compatible conditions to leave aminimal chemical residue. Cleavage of this probe from aDNA substrate resulted in abolition of the PCR bias effect.The application of chemical tagging approaches to genomicquestions has yielded powerful methods, and will certainlycontinue to feature in our study of the genome. However, wemust not neglect the effects on downstream processes such asPCR amplification and sequencing in our experimental designand analysis.GRM is supported by Trinity College and Herchel Smithstudentships. EAR is a Herchel Smith Fellow. The SB lab issupported by core funding from Cancer Research UK. SB is aWellcome Trust Senior Investigator.Notes and references1 A. M. Deaton and A. Bird, Genes Dev., 2011, 25, 1010–1022.2 S. Suzuki, R. Ono, T. Narita, A. J. Pask, G. Shaw, C. Wang, T. Kohda,A. E. Alsop, J. A. Marshall Graves, Y. Kohara, F. Ishino, M. B. Renfreeand T. Kaneko-Ishino, PLoS Genet., 2007, 3, e55.3 R. M. Kohli and Y. Zhang, Nature, 2013, 502, 472–479.4 M. Wossidlo, T. Nakamura, K. Lepikhov, C. J. Marques,V. Zakhartchenko, M. Boiani, J. Arand, T. Nakano, W. Reik andJ. Walter, Nat. Commun., 2011, 2, 241.5 W. A. Pastor, Y. Huang, H. R. Henderson, S. Agarwal and A. Rao, Nat.Protoc., 2012, 7, 1909–1917.6 C.-X. Song, K. E. Szulwach, Y. Fu, Q. Dai, C. Yi, X. Li, Y. Li,C.-H. Chen, W. Zhang, X. Jian, J. Wang, L. Zhang, T. J. Looney,B. Zhang, L. a. Godley, L. M. Hicks, B. T. Lahn, P. Jin and C. He, Nat.Biotechnol., 2011, 29, 68–72.7 M. Yu, G. C. Hon, K. E. Szulwach, C.-X. Song, P. Jin, B. Ren andC. He, Nat. Protoc., 2012, 7, 2159–2170.8 E.-A. Raiber, D. Beraldi, G. Ficz, H. E. Burgess, M. R. Branco,P. Murat, D. Oxley, M. J. Booth, W. Reik and S. Balasubramanian,Genome Biol., 2012, 13, R69.9 C.-X. Song, K. E. Szulwach, Q. Dai, Y. Fu, S.-Q. Mao, L. Lin, C. Street,Y. Li, M. Poidevin, H. Wu, J. Gao, P. Liu, L. Li, G.-L. Xu, P. Jin andC. He, Cell, 2013, 153, 678–691.10 X. Lu, C. Song, K. Szulwach, Z. Wang, P. Weidenbacher, P. Jin andC. He, J. Am. Chem. Soc., 2013, 135, 9315–9317.11 S. Ito, L. Shen, Q. Dai, S. C. Wu, L. B. Collins, J. A. Swenberg, C. Heand Y. Zhang, Science, 2011, 333, 1300–1303.12 D. Aird, M. G. Ross, W.-S. Chen, M. Danielsson, T. Fennell, C. Russ,D. B. Jaffe, C. Nusbaum and A. Gnirke, Genome Biol., 2011, 12, R18.13 P. Aller, M. A. Rould, M. Hogg, S. S. Wallace and S. Doublié, Proc.Natl. Acad. Sci. U. S. A., 2007, 104, 814–818.14 Y. Huang, W. A. Pastor, Y. Shen, M. Tahiliani, D. R. Liu and A. Rao,PLoS One, 2010, 5, e8888.15 D. R. Bentley, et al., Nature, 2008, 456, 53–59.16 K. Kubo, H. Ide, S. S. Wallace and Y. W. Kow, Biochemistry, 1992, 31,3703–3708.17 K. Kirouac, A. Basu and H. Ling, J. Mol. Biol., 2013, 425, 4167–4176.18 H. Hashimoto, S. Hong, A. S. Bhagwat, X. Zhang and X. Cheng,Nucleic Acids Res., 2012, 40, 10203–10214.Fig. 3 A qPCR study showing the relationship between DNA-adduct char-acter, prevalence, and inhibition of PCR. DCt is relative to a 5fC-containingtemplate. Bars show the average of three experiments, each performed intechnical triplicate; error bars show the S.E.M. Statistical significance wascalculated using two-way ANOVA (*P o 0.001, **P o 0.0001).Fig. 4 Templates containing five modification sites between positions fiftyand sixty were templates for a primer extension assay: (a) no modification(b) 5fC (c) DNA-1 or (d) DNA-1 cleavage products. (A) Schematic showingprimer extension along a template (black) to give a product (red). Chemicaltagging inhibits polymerase action and yields truncated products; probecleavage rescues this effect. (B) Denaturing PAGE shows probe-inducedtruncation products at modified sites, and the rescue achieved afterchemical cleavage to yield near-natural DNA.ChemComm CommunicationOpen Access Article. Published on 18 August 2014. Downloaded on 04/09/2015 10:07:54.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Chemical biology of genomic DNA: minimizing PCR bias.

https://www.repository.cam.ac.uk/bitstream/1810/245848/3/McInroy%20et%20al%202014%20Chemical%20Communications.pdf

Chemical biology of genomic DNA: minimizing PCR bias.

Abstract

Similar works

Full text

Available Versions

Apollo (Cambridge)