Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes

Sensitive and selective amplification of methylated DNA sequences using helper-dependent chain reaction in combination with a methylation-dependent restriction enzymes
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  Sensitive and selective amplification of methylatedDNA sequences using helper-dependent chainreaction in combination with a methylation-dependent restriction enzymes Keith N. Rand 1, *, Graeme P. Young 2 , Thu Ho 1 and Peter L. Molloy  1 1 CSIRO Animal, Food and Health Sciences, Preventative Health Flagship, North Ryde, NSW 1670,  2 FlindersCentre for Cancer Prevention and Control, Flinders University of South Australia, Adelaide, SA, Australia Received December 22, 2011; Revised July 18, 2012; Accepted August 9, 2012  ABSTRACTWe have developed a novel technique for specificamplification of rare methylated DNA fragments ina high background of unmethylated sequences thatavoids the need of bisulphite conversion. Themethylation-dependent restriction enzyme GlaI isused to selectively cut methylated DNA. Thentargeted fragments are tagged using specially designed ‘helper’ oligonucleotides that are alsoused to maintain selection in subsequent amplifica-tion cycles in a process called ‘helper-dependentchain reaction’. The process uses disabled primerscalled ‘drivers’ that can only prime on each cycle ifthe helpers recognize specific sequences withinthe target amplicon. In this way, selection for thesequence of interest is maintained throughout theamplification, preventing amplification of unwantedsequences. Here we show how the method can beapplied to methylated Septin 9, a promising bio-marker for early diagnosis of colorectal cancer.The GlaI digestion and subsequent amplificationcan all be done in a single tube. A detection sensi-tivity of 0.1% methylated DNA in a background ofunmethylated DNA was achieved, which wassimilar to the well-established Heavy Methylmethod that requires bisulphite-treated DNA.INTRODUCTION The fact that hypermethylation of tumour-associatedgenes frequently occurs in early stages of cancer develop-ment can be exploited for development of epigenetic bio-markers for early diagnosis, monitoring andprognostication of cancer (1–3). Indeed, the potential of several such biomarkers for early detection of cancer, forpredicting outcome and response to therapy and ultim-ately, for improved management of patients and thedisease has been clearly shown in translational andclinical settings (3). Examples include detection of hypermethylated (i) glutathione  S  -transferase (GSTP1)in urine and serum samples to help distinguish benignlesions from prostate cancer (4–6), (ii) vimentin 1 (7,8)in faeces and Septin 9 genes in blood to detect coloncancer (9) and (iii) multiple genes (including P16) insputum to mark early stages of lung cancer (10–13).Finally, clinical success of   O 6 -methylguanine-DNAmethyltransferase promoter hypermethylation as astrong prognostic factor for patients with newly diagnosedglioblastoma and as potent predictor of response to treat-ment with alkylating agents is a testament to the clinicalfeasibility of these concepts (14–19).There is now much interest in the use of CpG islandhypermethylation as a tool to detect cancer in the easilyaccessible body fluids and tissue specimens from cancerpatients (20). The issue with such type of samples,however, is the difficulty of detection of thetumour-derived DNA as it is generally present in lowquantities ( < 0.1%) (21) with most of the DNA beingderived from healthy cells. Thus, translational success of these approaches hinges on use of excellent detection tech-nology to detect minute amounts of rare methylated targetDNA in the presence of a vast excess of non-target DNAthat can include both related unmethylated andmethylated sequences.Several PCR-based assays have been developed, themajority of which depend upon the use of DNA treatedwith sodium bisulphite. Bisulphite treatment deaminatescytosine, but not 5-methyl cytosine, to uracil (22). Thisallows the preservation of the methylation informationthrough PCR which otherwise would not distinguishbetween methylated and non-methylated residues.Methylation-specific PCR (MSP) is the most widely usedapproach and yields excellent analytical sensitivity, *To whom correspondence should be addressed. Tel: +61 2 9490 5001; Fax: +61 2 9490 5020; Email: Published online 10 September 2012 Nucleic Acids Research, 2013, Vol. 41, No. 1  e15 doi:10.1093/nar/gks831   The Author(s) 2012. Published by Oxford University Press.This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the srcinal work is properly cited.  detecting up to 0.1% of methylated template in an excessof unmethylated DNA (23). Based on the differences inthe DNA sequences after the bisulphite treatment, theprimers are specially designed to amplify the methylatedtemplate only. The primary issue with this technology isthe potential for false positive due to mispriming or due toincomplete bisulphite conversion of DNA (24,25). In anattempt to tackle these issues, heavy methyl and headloopsuppression PCR methods, which also used bisulphite-treated DNA as the template, but maintain selectionagainst unmethylated sequences during each PCR cycle,were developed. Yielding comparable sensitivity to MSP,the ‘heavy methyl’ method minimizes the frequency of false positives through use of oligonucleotide blockersthat block amplification of unmethylated DNA (26). Inthe case of headloop suppression PCR, primers aredesigned to specifically suppress amplification of theunmethylated sequence (27).A particular example demonstrating the clinical feasi-bility of such methods is the application of the heavymethyl PCR method to detect methylated Septin 9 forearly diagnosis of colorectal cancer (CRC). Detection of methylation in the Septin 9 promoter region was found toeffectively discriminate CRC from normal specimens (28).The ability to detect circulating methylated Septin 9 DNAin plasma has led to the development of a minimallyinvasive blood test for CRC (9,29). As mentionedearlier, bisulphite-treated DNA is most commonly usedfor most detection assays. However, these assays sufferfrom the drawbacks of this technology, for example(i) preparation of bisulphite-treated DNA requiresmultiple steps (denaturation with NaOH, incubationwith Na-bisulphite, desulphonation and DNA purifica-tion) and hence, it is time-consuming; (ii) loss of material due to DNA degradation (30) and handlingsteps and (iii) suboptimal conversion rate based on tech-nical variability, DNA sequence, quality and samplesource which could sometimes lead to inconsistencies(31); this might be particularly relevant to clinicalspecimens.As an alternative to bisulphite treatment, restrictionenzyme (RE)-based methods have also been used forsite-specific detection of DNA methylation (32); basedon the type of RE, these enrich for either methylated orunmethylated DNA. Methylation-sensitive enzymes (e.g.HpaII, BstUI or AciI) are generally used and these cutonly if the DNA is unmethylated; the lack of cutting isthen detected by PCR of the methylated sequence usingflanking primers. The main source of error here is thepossibility of high background (false positives) arisingfrom incomplete cutting of the target sites and theapproach has not found wide acceptance. We haverecently described a RE-based method, end-specific PCR(ESPCR), which was used for detection of hypomethylation of repeated DNA sequences followingdigestion with methylation-sensitive enzymes (33).ESPCR uses specially designed helper or facilitator oligo-nucleotides to specifically target the cut fragments andthus is much less affected by incomplete digestion.The discovery of methylation-dependent enzymes thatcut at specific target sequences only if those sequences aremethylated allows this approach to be extended to thedetection of hypermethylation. Based on this premise, inthis proof of principle study and using Septin 9 as anexample, we describe a new methylation detectionmethod that avoids the use of bisulphite. After digestionwith the methylation-dependent enzyme GlaI (34,35), aparticular methylated Septin 9 fragment is specificallyamplified by using a new method called ‘helper-dependentchain reaction’ or ‘HDCR’. This combines ESPCR (33)with a unique feature that allows maintenance of the se-lection for the target sequence throughout amplificationand thus results in enhanced specificity. To our know-ledge, this is the first report of a method that results inthe continuous positive selection for a targeted sequence. MATERIALS AND METHODS DNAs and oligonucleotides K562 DNA, purified from a subculture of the humanchronic myelogenous leukaemia cell line, was obtainedfrom Promega. CpGenome Universal Methylated DNAis fully CpG-methylated human male genomic DNA andwas srcinally obtained from Chemicon (Temecula, CA,USA). It is now available from Millipore ( Tumour sampleswere obtained from patients undergoing surgery atFlinders Medical Centre (Adelaide, Australia) withconsent having been obtained prior to surgery—HumanResearch Ethics Committee approval RGH 09/04.Following tissue disruption using the Retsch TissueLyser(Qiagen), DNA from cancer and matched normal tissuewas isolated using a Wizard  Genomic DNA purificationKit (Promega).Oligonucleotide sequences are shown in Table 1. Thehelper oligonucleotides (denoted as ‘helper/s’ from hereon) were purchased from Biosearch Technologies(Novato, CA, USA) or from GeneWorks (Adelaide,Australia). Other oligonucleotides were purchased fromGeneWorks or from Sigma-Aldrich (Sydney, Australia).All oligonucleotides were dissolved in TEX buffer (10mMTris–HCl, 0.1mM EDTA, 0.01% Triton X-100). Heavy methyl PCR assay Genomic DNAs were bisulphite-converted using the EZDNA Methylation-Gold TM kit (Zymo Research). TheSeptin 9 heavy methyl assay was done, using QuantitectPCR mix without ROX (Qiagen) as described by de Vos2009 (9). GlaI digestion GlaI RE was purchased from Sibenzyme ( DNAs pre-cut with GlaIwere prepared by digesting 1 m g in ‘SE Buffer GlaI’(10mM Tris–HCl, pH 8.5 at 25  C; 5mM MgCl 2 ; 10mMNaCl; 1mM 2-mercaptoethanol) plus 16 units of GlaI in atotal volume of 50 m l for 2h at 30  C followed by 15min at70  C. After adding 5 m l of 50mM EDTA and 145 m l of TEX, the 5ng/ m l digested DNAs were stored at   20  C. e15  Nucleic Acids Research, 2013,Vol. 41,No. 1  P AGE  2 OF  10  Helper-dependent chain reaction HDCR was carried out using a Corbett Rotor-Gene 3000machine with a 72-tube rotor and 10 m l volumes.Conditions for hot start PCR were 20mM Tris–HCl(pH 8.4), 50mM KCl, 7.5mM MgCl 2 , 0.2mM dNTPs,oligonucleotides (as listed in Table 1), 1/50000 dilutionof SYBR Green, 0.01mM dithiothreitol, 0.1% TritonX-100 and 0.04U/ m l Platinum Taq Polymerase. GlaIwas included at 0.04units/ m l when the restriction digestwas carried out in the PCR tube.Suitable cycling conditions for HDCR for 10 m l volumesand the Rotor-Gene 3000 using pre-cut DNA sampleswere five cycles (90  C 15 s, 55  C 20 s, 76  C 5 s, 67  C 20s) followed by second stage of up to 85 cycles (90  C 15s— read HEX channel, 67  C 40 s, 76  C 5 s, 67  C 20 s—readFAM/SYBR Green channel). For the results shown inFigure 3B, 50  C was used in place of 55  C in the firststage. When GlaI digestion was done in the PCR mix,an initial incubation of 10min at 30  C was carried out.The SYBR Green is included to allow a more completeanalysis of the amplification if necessary, for example byallowing a melt curve analysis to be carried out after theamplification. We have found that the addition of a lowlevel of Triton X-100 in some situations improves theSYBR Green signal; possibly by preventing loss of thehydrophobic SYBR Green to surfaces. The dithiothreitolappears in our experience to be needed to stabilize somebatches of Platinum Taq Polymerase, especially when highdenaturation temperatures are used. For quantification,all samples were diluted such that 2ng of each wasassayed with a standard curve ranging from 100pg to2ng of fully methylated CpGenome DNA (mixed withK562 DNA in which Septin 9 is unmethylated to makea total amount of 2ng, where necessary). The standardswere cut by GlaI in the initial incubation of the PCR tubesat 30  C, along with the test samples. The correlationbetween the two assays was done by assessment of Spearman’s rank correlation coefficient (Spearman’s rho)using the Graph Pad Prism, version 5. RESULTS We describe here a new one-step PCR-based HDCRmethod that can be used to detect cancer-associatedhypermethylation in clinical samples. To show sensitivityand specificity of the assay, we used commercially avail-able DNAs so that other laboratories will be able to makedirect comparisons with other assays already in use. The concept and strategy Overall, three primary steps are involved: (1) genomicDNA is cut with the methylation-dependent RE GlaI,(2) the GlaI-cut fragment from the target gene is specific-ally tagged with 3 0 -extensions at each cut end and (3)HDCR is used to specifically amplify and detect thetagged molecules that derive only from the methylatedtarget. In these experiments, the Septin 9 gene that ishypermethylated in  > 90% of CRC patients (29) wastargeted. Generation of a methylation-dependent restriction digest Genomic DNA is cut with the GlaI RE; GlaI cleaves theDNA sequence, 5 0 -GCGC-3 0 /3 0 -CGCG-5 0 if there are two,three or four 5-methylcytosines (5meC, or ‘M’) (34,35)(Figure 1A). The efficiency of cleavage is variable(6%–100%) based on the quantity and location of 5meCs, as well as the composition of the enzyme recogni-tion sequence. Fragments generated will be derived fromDNA that is normally methylated as well as sequencesthat are specifically methylated in cancer cells [e.g.GlaI-cut Septin 9 gene fragment (Figure 1A)]. Thehypermethylated region in the Septin 9 gene includestwo consensus GlaI target sites that are expected to yielda fragment of 45bp after GlaI digestion (Figure 1Band C). Tagging of GlaI-cut candidate gene using gene-specific‘helper’ oligonucleotide In this step, the target hypermethylated gene is taggedusing a specially designed oligonucleotide—called a‘helper’, which can bind but cannot be extended thus pre-venting any copying of annealed genomic DNA.Specifically, the helper contains at its 5 0 -end a non-gene-specific tag sequence (TAG) followed by a gene-specificsequence and then a blocked 3 0 -end. The 3 0 -end comprisesa stretch of three 2 0 - O -methyl residues adjacent to a3 0 -nucleotide that is mispaired with the target sequence.The gene-specific elements of the helper lead to specifichybridization to the target. If the target has been cut byGlaI, then it becomes tagged by copying of the non-gene-specific 5 0 -helper sequences (Figure 1C). HDCR detects the hypermethylated candidate genes inGlaI-cut DNA mix HDCR is designed to increase the specificity of detectionthrough use of a specially designed primer—called the‘driver’ together with the ‘helper’ (Figure 2). Table 1.  Oligonucleotide sequences and final concentrations used in Septin 9 GlaI-HDCR assayOligo name Role Sequence 5 0 to 3 0 Purification and source Final conc. (nM)UDr2H Driver F CCCGTMGMMGTMMMTMuuuMTAMAG HPLC, GeneWorks 400UDr3H Driver R CGCCTMMMGMMTGMTMuuuMATMGA HPLC, GeneWorks 400S9m3HF354 Helper F GTCGCCGTCCCTCTTTCTACAGTGcgaCCCGCTGCCCuuuT PCR grade, GeneWorks 25S9m3HR355 Helper R CTCCCGCCTGCTCTTTCATCGATGuugACCGCGGGGTCCuuuT PCR grade, GeneWorks 25S9HEXPr Probe HEX- CAC[+C]AG[+C]C[+A]T[+C]AT[+G]TCG BHQ1 HPLC, Sigma 50F=‘Forward’, R=‘Reverse’; 2 0 - O -methyl nucleotides are shown in lower case; 5-methyl cytosines are shown as ‘M’; locked nucleic acid residues areshown as [+C]. P AGE  3 OF  10  Nucleic AcidsResearch, 2013, Vol.41,No. 1  e15  The driver is complementary to the extended TAGsequence and acts as a primer to copy the target GlaIfragment of the Septin 9 sequence and extension of thetarget strand occurs (Figure 2, Step 1). However, in thesecond strand synthesis, the block in the driver causes pre-mature termination and prevents the regeneration of thefull binding site for the driver (Figure 2, Step 2). Here theblock to extension is caused by the presence of three 2 0 - O -methyl uracil bases in the driver that cause the polymeraseto stall.Amplification then becomes dependent upon the‘helper’ that is required to allow completion of strand syn-thesis. As in the initial tagging reaction (Figure 1), thehelper anneals to the 3 0 -end of the incomplete strand,the Septin 9 sequences and first six (underlined in Steps2 and 3 in Figure 2) bases of the TAG sequence. Specificbinding leads to the copying of the 5 0 tail of the helper,thus regenerating the binding site for the driver (Figure 2,Step 3).After denaturation, the driver binds to the regeneratedsite and the amplification cycle continues (Figure 2, Step4). Because continued specific binding of the helper to thetarget gene sequence is required through the entire ampli-fication, very high specificity is potentially achieved.As is evident in Figure 2, the sequence of the driverprimer is included within that of the helperoligonucleotide and both will compete for hybridizationto the tagged Septin 9 strand. In order to improve ampli-fication efficiency, sequences and reaction conditions needto be optimized to favour the driver binding and priming.Here we have increased the driver’s hybridization stability(i.e.  T  m  value) through replacement of some of the cyto-sines by 5MeC in its sequence. This has been shown toincrease stability of binding in other systems (36,37).Additional bias towards driver binding was obtainedthrough keeping the concentration of the helper oligo-nucleotide much lower (40nM) relative to the driver(400nM). Furthermore, after Step 2 (Figure 2) a tempera-ture spike to 76  C is included to facilitate replacement of the helper with the driver and permit priming of the nextround of amplification. HDCR and detection of hypermethylated Septin 9 The Septin 9 HDCR assay uses a helper/driver combin-ation at both ends of the 45bp GlaI Septin 9 fragment(Table 1). In Figure 2, for clarity, the scheme only showsreactions from one end. Successful amplification of Septin9 was shown by using a specific HEX-labelled TaqMan  probe (Table 1). The performance of the assay was ini-tially demonstrated using CpGenome fully methylatedDNA and K562 DNA as a control in which the Septin 9gene is unmethylated. To investigate the specificity of the Figure 1.  Methylation-dependent restriction of genomic DNA and sequence-specific 3 0 -0 tagging of the cut fragment using the helper. Panel (A): aschematic describing the methylation-dependent enzyme (GlaI)-based restriction of normal genomic DNA (fragments shown in blue) or that from thecancer patients (fragments shown in solid black). The fragment representing methylated Septin 9 is shown. Panel (B): the cartoon shows the GlaIconsensus sites and a schematic of Septin 9 digestion using GlaI generating a 45-bp sequence; M: 5-methylcytosine. Panel (C): after GlaI digestion thecut fragment (example of Septin 9 sequences is shown) is then tagged at its 3 0 -ends using two ‘helpers’ (shown in blue). The helper shown in the upperpart of the figure contains a non-gene-specific ‘Tag’ sequence followed by a gene-specific sequence and a 3 0 -sequence uuuT (u=2 0 - O -methyl uraciland T is a mismatch with the target) designed to prevent extension. A second helper with similar features is used to tag the other 3 0 -end. Theresulting tags on the 3 0 -ends of both strands of the target Septin 9 target are shown in green. e15  Nucleic Acids Research, 2013,Vol. 41,No. 1  P AGE  4 OF  10  reaction, we have studied various mixtures of DNAs(Figure 3). The sensitivity of HDCR was determinedusing samples containing different concentrations of methylated DNA pre-cut with GlaI (10pg–10ng); thehypermethylation in the Septin 9 gene could be detectedin samples containing only 10pg of GlaI-cut genomicDNA (Figure 3A). Because driver hybridization andpriming is reduced due to competition from the helper,the efficiency of amplification is significantly reducedcompared with standard PCR and detection requiresmore cycles. Nevertheless, the requirement for specific an-nealing with sequences internal to the Septin 9 fragmentduring each cycle leads to a prolonged suppression of background amplification. Specificity was furtherexamined in DNA mixtures containing different propor-tions of GlaI-pre-cut methylated DNA (0 or 0.1% or100%). HDCR could accurately detect methylatedSeptin 9 in samples containing only 0.1% of methylatedDNA (Figure 3B). HDCR of the hypermethylated Septin 9 gene in clinicalsamples As proof of principle, HDCR was employed to evaluatethe extent of hypermethylated Septin 9 gene in CRC andmatched normal tissues from 25 cancer patients (fourStage A, nine Stage B, eight Stage C, one Stage D andfour unknown stage). The level of methylation wasdetermined relative to that of fully methylatedCpGenome DNA. A clear separation of cancer andnormal tissue DNA methylation is evident (Figure 4A).In considering individual samples, HDCR could detecthypermethylation in the Septin 9 gene to a level of   > 5%in 22/25 (88%) patients (Figure 4B). For all but two of thesubjects, methylation of the Septin 9 gene was Figure 2.  HDCR: quantitative PCR-based selective amplification of tagged Septin 9. This figure describes the main features of HDCR that areneeded to maintain selection for the correct target throughout amplification. (1) For the sake of clarity, reactions are shown for one strand only.Only the lower, 3 0 - to 5 0 -tagged Septin 9 strand from Figure 1 is followed here. (2) A specially designed oligonucleotide, the ‘driver’ (blue sequence),anneals to the tag (green). The driver contains three 2 0 - O -methyl nucleotides but because they are located 6nt from the 3 0 -end, they do not preventextension and thus the copying of the Septin 9 fragment. (3) In the next round of replication, the new strand formation (copying) stops at the ‘block’sequence (uuu) in the driver, thus preventing the regeneration of the driver-binding site. (4) After denaturation, an oligonucleotide called the ‘helper’(shown in red) hybridizes specifically to the prematurely terminated strand. The hybridizing site consists of 7nt of tag sequence plus 14nt of Septin 9.The helper contains two stretches of 2 0 - O -methyl nucleotides. The stretch close to the helper’s 3 0 -end (lower case, uuu), in combination with a3 0 -terminal mismatch (T/T), is designed to prevent extension of the helper. The centrally located stretch of 2 0 - O -methyl nucleotides (lower case, cga) isnot essential to the HDCR process. It is designed to reduce background that might occur due to inappropriate priming of non-target fragments onthe helper. Because the helper shares 5 0 -sequences with the driver, copying of the helper’s 5 0 -tail regenerates the driver’s binding site. (5) A 76  Ctemperature spike is sufficient to remove the helper used here, allowing the driver to access its regenerated binding site. It is important to note that atthis step the driver will be in competition with the helper for binding. Modifications that decrease helper  T  m  or that increase driver  T  m  (such as theuse of 5MeC as used here) and the use of a 10-fold higher concentration of driver aids binding and thus copying of the Septin 9 strand. Selection forthe correct product is maintained throughout the amplification because of the continued need of specific helper binding to the Septin 9 sequence inevery cycle. P AGE  5 OF  10  Nucleic AcidsResearch, 2013, Vol.41,No. 1  e15
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