Methylated Templates Research Articles

Specialized replication of heterochromatin domains ensures self-templated chromatin assembly and epigenetic inheritance

Heterochromatin, defined by histone H3 lysine 9 methylation (H3K9me), spreads across large domains and can be epigenetically inherited in a self-propagating manner. Heterochromatin propagation depends upon a read-write mechanism, where the Clr4/Suv39h methyltransferase binds to preexisting trimethylated H3K9 (H3K9me3) and further deposits H3K9me. How the parental methylated histone template is preserved during DNA replication is not well understood. Here, we demonstrate using Schizosaccharomyces pombe that heterochromatic regions are specialized replication domains demarcated by their surrounding boundary elements. DNA replication throughout these domains is distinguished by an abundance of replisome components and is coordinated by Swi6/HP1. Although mutations in the replicative helicase subunit Mcm2 that affect histone binding impede the maintenance of a heterochromatin domain at an artificially targeted ectopic site, they have only a modest impact on heterochromatin propagation via the read-write mechanism at an endogenous site. Instead, our findings suggest a crucial role for the replication factor Mcl1 in retaining parental histones and promoting heterochromatin propagation via a mechanism involving the histone chaperone FACT. Engagement of FACT with heterochromatin requires boundary elements, which position the heterochromatic domain at the nuclear peripheral subdomain enriched for heterochromatin factors. Our findings highlight the importance of replisome components and boundary elements in creating a specialized environment for the retention of parental methylated histones, which facilitates epigenetic inheritance of heterochromatin.

Methylation levels assessment with Methylation-Sensitive High-Resolution Melting (MS-HRM).

Testing for disease-related DNA methylation changes provides clinically relevant information in personalized patient care. Methylation-Sensitive High-Resolution Melting (MS-HRM) is a method used for measuring methylation changes and has already been used in diagnostic settings. This method utilizes one set of primers that initiate the amplification of both methylated and non-methylated templates. Therefore, the quantification of the methylation levels using MS-HRM is hampered by the PCR bias phenomenon. Some approaches have been proposed to calculate the methylation level of samples using the high-resolution melting (HRM) curves. However, limitations of the methylation calculation using MS-HRM have not been evaluated systematically and comprehensively. We used the Area Under the Curve (AUC), a derivative of the HRM curves, and least square approximation (LSA) to establish a procedure that allowed us to infer methylation levels in an MS-HRM experiment and assess the limitations of that procedure for the assays’ specific methylation level measurement. The developed procedure allowed, with certain limitations, estimation of the methylation levels using HRM curves.

Sensitive lateral flow assay for bisulfite-free DNA methylation detection based on the restriction endonuclease GlaI and rolling circle amplification

The detection of DNA methylation with high sensitivity and specificity is important for the early diagnosis of many human diseases, including cancers. Here, we integrated the high specificity of the methylation-dependent restriction endonuclease GlaI for methylation-dependent digestion and the high amplification efficiency of rolling circle amplification (RCA) for the detection of GlaI digestion products. GlaI can only digest a methylated template, leading to the generation of digestion products with specific ends. The specific digestion product can then be ligated to a ligation mediator with a dumbbell structure to generate a complete circular template for further RCA, and the final RCA amplicon can be detected using lateral flow detection (LFD) with the naked eye. The specificity of Gla-RCA not only depends on the specific methylation digestion of GlaI, but only the ligation process of RCA amplification. As a proof of principle, the sensitivity of GlaI-RCA assay was applied to methylated Septin 9 and showed a sensitivity of approximately 1% (50 copies of methylated template per reaction) and no cross-reactivity with 5000 copies of unmethylated DNA used as background. The application of GlaI-RCA was also evaluated with colorectal cancer tissue samples and showed great accordance with standard bisulfite sequencing. A bisulfite-free and LFD-based DNA methylation detection was successfully developed, promising high specificity and rapid visual detection and having a great potential to become a robust tool for DNA methylations analysis.

CRISPR/Cas9-mediated epigenetic editing tool: An optimized strategy for targeting de novo DNA methylation with stable status via homology directed repair pathway

With the development of epigenetic engineering tools for manipulating DNA methylation at the desired locus, the stable maintenance of targeted methylation status in edited cells after division poses a major obstacle. Transient methylation levels, along with the reversible inhibition of the desired genes, by some epigenetic editing techniques, limit their further application in functional and therapeutic studies of critical regulators in the cancer epigenome. In this study, we utilized CRISPR/Cas9-mediated the homology-directed repair (HDR) pathway following double-strand breaks (DSBs) information to target de novo methylation of the vital epigenetic biomarker, O6-methylguanine-DNA methyltransferase (MGMT). Based on synthetic single and double stranded methylated repair templates, DNA methylation patterns can be incorporated into the endogenous MGMT gene. In addition, we observed upregulation of DNA methyltransferases (DNMTs) in edited HeLa cells. In particular, we employed an optimized method of using the DNA ligase IV inhibitor SCR7 to markedly enhance the insertion efficiency induced by the long methylated repair template, contributing to the induction of stable methylation alterations through epigenetic recombination after cell division. Additionally, our study confirmed that CRISPR/Cas9-mediated target methylation in a site-specific manner enabled the maintenance of gene silencing in vitro and in vivo. Collectively, we show that a combination of CRISPR/Cas9 components, SCR7 treatment, and the long methylated HDR template can enhance CRISPR/Cas9-directed epigenomic editing efficiency and further induce stable effects on methylation modifications and transcriptional suppression.

Optimized inverse PCR strategy for constructing multilocus mutants efficiently

Mutants of proteins are the basis for studying their structure and function, this work aimed to establish an efficient and rapid method for constructing multi-site mutants. When four or more adjacent amino acid residues need to be mutated, firstly, two long and two short primers (long primers Ⅰ/Ⅰ, short primersⅡ/Ⅱ) were designed: the long primers contain mutated sites, and the number of mutant bases is ≤20 bp, the short primers do not contain mutated sites; GC contents of the long and short primers are ≤80%, and the difference of annealing temperature is ≤40 °C. Then two sets of reverse PCR amplifications were performed using primer pairs (Ⅰ/Ⅱand Ⅰ/Ⅱ) and templates, respectively. After amplification, each system can obtain non-methylated linear plasmids which contain mutated sites, and the breakpoints of the two sets of linear plasmids amplified by primers Ⅰ/Ⅱ and Ⅲ/Ⅳ were distributed on both sides of the mutated sites. Followed by digested by DpnⅠ to remove the methylated templates, the recovered PCR products, which were mixed in an equimolar ratio, were performed another round of denaturation and annealing: the two sets of linear plasmids were denatured at 95 °C and then annealed with each other's single-stranded DNA as templates to form open-loop plasmids, and then the transformants containing the mutations will be obtained after transformed the open-loop plasmids into Escherichia coli competent cells. Results showed that, this method can mutate 4 to 11 consecutive amino acid residues (8-20 bp) simultaneously, which will greatly simplify the construction of multi-site mutants, Thereby improve the efficiency of protein structure and function research further.

Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5′ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Methylated Cytosine Maintains G-Quadruplex Structures during Polymerase Chain Reaction and Contributes to Allelic Dropout.

The promoter of the human imprinted gene MEST is differentially methylated with respect to the parent of origin and contains several non B-DNA motifs that are capable of forming G-quadruplexes. These factors can contribute to a consistent allelic dropout (ADO) of the maternally methylated DNA during polymerase chain reaction (PCR) analysis of such gene regions. Here, we directly investigate the cause of allelic dropout by applying fluorescent techniques to visualize polymerase amplification and arrest during PCR of differentially methylated DNA templates. We demonstrate that polymerase arrest corresponds to previously characterized G-quadruplex-forming motifs at the MEST promoter region and occurs at equivalent sites on both methylated and nonmethylated DNA templates. However, during PCR, polymerase arrest can be observed on the methylated template for several cycles longer than on the nonmethylated template, and this results in an amplification lag and a lower yield of full length amplicons. We demonstrate that this delay in amplification is sufficient to cause complete ADO during PCR, providing a mechanistic basis for the previously observed genotyping error at this locus.

Evaluation of Different Oligonucleotide Base Substitutions at CpG Binding sites in Multiplex Bisulfite-PCR sequencing

Multiplex bisulfite-PCR sequencing is a convenient and scalable method for the quantitative determination of the methylation state of target DNA regions. A challenge of this application is the presence of CpGs in the same region where primers are being placed. A common solution to the presence of CpGs within a primer-binding region is to substitute a base degeneracy at the cytosine position. However, the efficacy of different substitutions and the extent to which bias towards methylated or unmethylated templates may occur has never been evaluated in bisulfite multiplex sequencing applications. In response, we examined the performance of four different primer substitutions at the cytosine position of CpG’s contained within the PCR primers. In this study, deoxyinosine-, 5-nitroindole-, mixed-base primers and primers with an abasic site were evaluated across a series of methylated controls. Primers that contained mixed- or deoxyinosine- base modifications performed most robustly. Mixed-base primers were further selected to determine the conditions that induce bias towards methylated templates. This identified an optimized set of conditions where the methylated state of bisulfite DNA templates can be accurately assessed using mixed-base primers, and expands the scope of bisulfite resequencing assays when working with challenging templates.

Manipulation of primer affinity improves high-resolution melting accuracy for imprinted genes.

High-resolution melting (HRM) is considered an inexpensive, rapid, and attractive methodology for methylation analysis. In the application of the polymerase chain reaction (PCR) to methylation analysis, amplification efficiencies are biased towards unmethylated, rather than methylated, templates: a phenomenon known as PCR bias. To overcome PCR bias, primers that include CpG site(s) and are fully complementary to the methylated sequence have been proposed. However, genes mapped within imprinted regions usually present higher methylation levels, and an unusual PCR bias towards the methylated template can therefore arise. The manipulation of primer affinity attempts to overcome this problem. We attempted to show that mismatches at the primer's methylated binding sites increase the area between the 50 and 100% methylation plots on the melting curves, and may increase HRM accuracy for samples that have high methylation levels. Sets of primers for imprinted genes that included CpG sites at their binding sequences were designed, and were complementary to methylated or unmethylated templates. Primers fully complementary to methylated templates produced a very small area between the 50 and 100% methylation plots. When using primers that were fully complementary to the unmethylated sequence, we were able to increase the area between the 50 and 100% methylation plots. Therefore, when samples are highly methylated, such as targets in genes mapped in imprinted regions, we propose that primers should favor amplification of the rarest, unmethylated sequence. Primers may be designed to include one CpG at its binding site and be fully complementary to the unmethylated template.

A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assembly

A hallmark of histone H3 lysine 9 (H3K9)-methylated heterochromatin, conserved from the fission yeast Schizosaccharomyces pombe to humans, is its ability to spread to adjacent genomic regions. Central to heterochromatin spread is heterochromatin protein 1 (HP1), which recognizes H3K9-methylated chromatin, oligomerizes and forms a versatile platform that participates in diverse nuclear functions, ranging from gene silencing to chromosome segregation. How HP1 proteins assemble on methylated nucleosomal templates and how the HP1-nucleosome complex achieves functional versatility remain poorly understood. Here we show that binding of the key S. pombe HP1 protein, Swi6, to methylated nucleosomes drives a switch from an auto-inhibited state to a spreading-competent state. In the auto-inhibited state, a histone-mimic sequence in one Swi6 monomer blocks methyl-mark recognition by the chromodomain of another monomer. Auto-inhibition is relieved by recognition of two template features, the H3K9 methyl mark and nucleosomal DNA. Cryo-electron-microscopy-based reconstruction of the Swi6-nucleosome complex provides the overall architecture of the spreading-competent state in which two unbound chromodomain sticky ends appear exposed. Disruption of the switch between the auto-inhibited and spreading-competent states disrupts heterochromatin assembly and gene silencing in vivo. These findings are reminiscent of other conditionally activated polymerization processes, such as actin nucleation, and open up a new class of regulatory mechanisms that operate on chromatin in vivo.

AIM1 and LINE-1 Epigenetic Aberrations in Tumor and Serum Relate to Melanoma Progression and Disease Outcome

Aberrations in the methylation status of non-coding genomic repeat DNA sequences and specific gene promoter region are important epigenetic events in melanoma progression. Promoter methylation status in LINE-1 and Absent in melanoma-1(AIM1;6q21) associated with melanoma progression and disease outcome was assessed. LINE-1 and AIM1 methylation status was assessed in paraffin-embedded archival tissues(PEAT)(n=133) and melanoma patients’ serum(n=56). LINE-1 U-Index(hypomethylation) and AIM1 were analyzed in microdissected melanoma PEAT sections. The LINE-1 U-Index of melanoma(n=100) was significantly higher than that of normal skin(n=14) and nevi(n=12)(P=0.0004). LINE-1 U-Index level was elevated with increasing AJCC stage(P<0.0001). AIM1 promoter hypermethylation was found in higher frequency(P=0.005) in metastatic melanoma(65%) than in primary melanomas(38%). When analyzed, high LINE-1 U-Index and/or AIM1 methylation in melanomas were associated with disease-free survival(DFS) and overall survival(OS) in Stage I/II patients (P=0.017, 0.027; respectively). In multivariate analysis, melanoma AIM1 methylation status was a significant prognostic factor of OS(P=0.032). Furthermore, serum unmethylated LINE-1 was at higher levels in both stage III(n=20) and stage IV(n=36) patients compared to healthy donors(n=14)(P=0.022). Circulating methylated AIM1 was detected in patients’ serum and was predictive of OS in Stage IV patients (P=0.009). LINE-1 hypomethylation and AIM1 hypermethylation have prognostic utility in both melanoma patients’ tumors and serum.

Abstract 3175: Detection of hypermethylated vimentin gene in urine of colorectal cancer patients

Abstract Although the success of cancer treatment often depends on the early detection of cancer, colorectal cancer (CRC) screening is hindered by low compliance rates (40%) of inconvenient, invasive screening tests. Previous studies have demonstrated that urine contains circulation-derived low-molecular-weight (LMW) DNA (&amp;lt;300 bp) and suggested that LMW urine DNA could be used to detect CRC associated genetic alterations. Aberrant hypermethylation of the vimentin gene (mVIM) has been shown to be a potential stool DNA marker for CRC screening. It was of interest to see whether mVIM could be detected in urine of patients with CRC. In this study, we developed a quantitative MethyLight assay targeting a 39 bp DNA template for mVIM. A locked nucleic acid was incorporated at the CpG sites of the reverse primer of the assay to increase the assay sensitivity and specificity for methylated templates. A blinded concordance study between urine and matched CRC tissue was performed. Twenty matched sets of urine and CRC tissue were collected and subjected to DNA isolation. Total urine DNA was further fractionated into both LMW DNA (&amp;lt; 1 kb) and high-molecular-weight (HMW) DNA (&amp;gt;1 kb) fractions. The MethyLight assay was used to quantify the amount of mVIM in each DNA sample. mVIM was detected in 75% (15/20) of the LMW urine DNA fraction, 0% (0/20) of the HMW urine DNA fraction and 85% (17/20) of the CRC tissue DNA. There was a 60% concordance in detecting mVIM between the CRC tissues and their corresponding urine samples. As negative controls, twenty LMW urine DNA fractions from subjects with no known neoplasia were also tested for mVIM and only 10% (2/20) of control samples contained detectable mVIM. mVIM may be a useful part of a urine based genetic biomarker panel for the early detection of CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3175. doi:10.1158/1538-7445.AM2011-3175

Chemogene treatment consisting of recombinant adenoviral transfection of p16cDNA (SVN-22/3), and docetaxel eradicates chemoresistant aneuploid pancreatic adenosquamous ca characterised by overexpression of k-ras and hypermethylation of CPG islands of P16

IntroductionAdenosquamous Ca is an aggressive and highly metastatic variant of adenocarcinoma with both glandular and squamous differentiation. Usually, it occurs in chemoradiated patients.MethodsTumour cells were obtained from a resected pancreatic...

Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies

The methylation-sensitive high-resolution melting (MS-HRM) protocol, as described by Wojdacz and Dobrovic, enables detection of a methylated template in an unmethylated background, with sensitivity similar to that of methylation-specific PCR (MSP). Furthermore, MS-HRM-based methylation screening is cost, labor and time efficient in contrast to direct bisulfite sequencing, which, therefore, is unsuitable as a screening method, but is still required to reveal the methylation status of individual CpG sites. In some experiments, detailed information on the methylation status of individual CpGs may be of interest for at least a subset of samples from MS-HRM-based methylation screening. For those samples, sequencing-based methodology has to be coupled with the MS-HRM protocol to investigate the methylation status of single CpG sites within the locus of interest. In this article, we review the limitations and advantages of MS-HRM and bisulfite sequencing protocols for single-locus methylation studies. Furthermore, we provide the insights into interpretation of the results obtained when a combination of the protocols is used for single-locus methylation studies.

Quantitation of DNA methylation by melt curve analysis

BackgroundMethylation of DNA is a common mechanism for silencing genes, and aberrant methylation is increasingly being implicated in many diseases such as cancer. There is a need for robust, inexpensive methods to quantitate methylation across a region containing a number of CpGs. We describe and validate a rapid, in-tube method to quantitate DNA methylation using the melt data obtained following amplification of bisulfite modified DNA in a real-time thermocycler.MethodsWe first describe a mathematical method to normalise the raw fluorescence data generated by heating the amplified bisulfite modified DNA. From this normalised data the temperatures at which melting begins and finishes can be calculated, which reflect the less and more methylated template molecules present respectively. Also the T50, the temperature at which half the amplicons are melted, which represents the summative methylation of all the CpGs in the template mixture, can be calculated. These parameters describe the methylation characteristics of the region amplified in the original sample.ResultsFor validation we used synthesized oligonucleotides and DNA from fresh cells and formalin fixed paraffin embedded tissue, each with known methylation. Using our quantitation we could distinguish between unmethylated, partially methylated and fully methylated oligonucleotides mixed in varying ratios. There was a linear relationship between T50 and the dilution of methylated into unmethylated DNA. We could quantitate the change in methylation over time in cell lines treated with the demethylating drug 5-aza-2'-deoxycytidine, and the differences in methylation associated with complete, clonal or no loss of MGMT expression in formalin fixed paraffin embedded tissues.ConclusionWe have validated a rapid, simple in-tube method to quantify methylation which is robust and reproducible, utilizes easily designed primers and does not need proprietary algorithms or software. The technique does not depend on any operator manipulation or interpretation of the melt curves, and is suitable for use in any laboratory with a real-time thermocycler. The parameters derived provide an objective description and quantitation of the methylation in a specimen, and can be used to for statistical comparisons of methylation between specimens.

TAF12 Recruits Gadd45a and the Nucleotide Excision Repair Complex to the Promoter of rRNA Genes Leading to Active DNA Demethylation

Many studies have detailed the repressive effects of DNA methylation on gene expression. However, the mechanisms that promote active demethylation are just beginning to emerge. Here, we show that methylation of the rDNA promoter is a dynamic and reversible process. Demethylation of rDNA is initiated by recruitment of Gadd45a (growth arrest and DNA damage inducible protein 45 alpha) to the rDNA promoter by TAF12, a TBP-associated factor that is contained in Pol I- and Pol II-specific TBP-TAF complexes. Once targeted to rDNA, Gadd45a triggers demethylation of promoter-proximal DNA by recruiting the nucleotide excision repair (NER) machinery to remove methylated cytosines. Knockdown of Gadd45a, XPA, XPG, XPF, or TAF12 or treatment with drugs that inhibit NER causes hypermethylation of rDNA, establishes heterochromatic histone marks, and impairs transcription. The results reveal a mechanism that recruits the DNA repair machinery to the promoter of active genes, keeping them in a hypomethylated state.

Methylation-sensitive high-resolution melting

The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.

NRPD1a and NRPD1b are required to maintain post‐transcriptional RNA silencing and RNA‐directed DNA methylation in Arabidopsis

In plants, both transcriptional (TGS) and post-transcriptional gene silencing (PTGS) can be self-reinforcing, and this allows maintenance of silencing once the initiator has been removed or suppressed. For TGS, this can be accomplished by the generation of small interfering RNAs (siRNAs) from methylated DNA templates by RNA polymerase IV (PolIV), RNA-dependent RNA polymerase 2 (RDR2), DICER-LIKE 3 (DCL3), and the RNA-directed DNA methylation (RdDM) machinery. Maintenance of PTGS requires RNA-dependent RNA polymerase 6 (RDR6), and may be associated with DNA methylation and transitive production of secondary siRNAs. In this work, mutants defective for the NRPD1a and NRPD1b alternative largest subunits of PolIV were tested for their ability to undergo RdDM, transitive RNA silencing and maintenance of PTGS. PTGS could be initiated in both nrpd1a and nrpd1b mutants, and this was associated with production of secondary siRNAs; silencing was not maintained however. nrpd1a mutants could support RdDM although this was lost upon reversal of silencing, as was methylation in rdr6 mutants. We conclude that components of the machinery that maintain TGS are required for maintenance of PTGS, and that RDR6 uses distinct templates in the initiation and maintenance phases of RNA silencing.

The Saccharomyces cerevisiae PHM8 Gene Encodes a Soluble Magnesium-dependent Lysophosphatidic Acid Phosphatase

Phosphate is the essential macronutrient required for the growth of all organisms. In Saccharomyces cerevisiae, phosphatases are up-regulated, and the level of lysophosphatidic acid (LPA) is drastically decreased under phosphate-starved conditions. The reduction in the LPA level is attributed to PHM8, a gene of unknown function. phm8Delta yeast showed a decreased LPA-hydrolyzing activity under phosphate-limiting conditions. Overexpression of PHM8 in yeast resulted in an increase in the LPA phosphatase activity in vivo. In vitro assays of the purified recombinant Phm8p revealed magnesium-dependent LPA phosphatase activity, with maximal activity at pH 6.5. The purified Phm8p did not hydrolyze any lipid phosphates other than LPA. In silico analysis suggest that Phm8p is a soluble protein with no transmembrane domain. Site-directed mutational studies revealed that aspartate residues in a DXDXT motif are important for the catalysis. These findings indicated that LPA plays a direct role in phosphate starvation. This is the first report of the identification and characterization of magnesium-dependent soluble LPA phosphatase.

Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection

DNA methylation changes that are recurrent in cancer have generated great interest as potential biomarkers for the early detection and monitoring of cancer. In such situations, essential information is missed if the methylation detection is purely qualitative. We describe a new probe-free quantitative methylation-specific PCR (MSP) assay that incorporates evaluation of the amplicon by high-resolution melting (HRM) analysis. Depending on amplicon design, different types of information can be obtained from the HRM analysis. Much of this information cannot be obtained by electrophoretic analysis. In particular, identification of false positives due to incomplete bisulphite conversion or false priming is possible. Heterogeneous methylation can also be distinguished from homogeneous methylation. As proof of principle, we have developed assays for the promoter regions of the CDH1, DAPK1, CDKN2A (p16INK4a) and RARB genes. We show that highly accurate quantification is possible in the range from 100% to 0.1% methylated template when 25 ng of bisulphite-modified DNA is used as a template for PCR. We have named this new approach to quantitative methylation detection, Sensitive Melting Analysis after Real Time (SMART)-MSP.