A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation
© Tierling et al; licensee BioMed Central Ltd. 2010
Received: 20 April 2010
Accepted: 2 June 2010
Published: 2 June 2010
DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation.
This combined method allows detection of 14 pg (that is, four to five genomic copies) of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng) of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2) and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer.
The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.
Changes in DNA methylation such as hypermethylation of tumor suppressor genes are regarded as early molecular events during cancer development. Such epigenetic changes are widely used as molecular markers in tumor cell diagnostics [1–3]. Selective and robust PCR-based screening methods for very early detection of abnormal tumor-specific methylation become increasingly important. In particular, methods to screen for the low abundance of aberrantly methylated tumor DNA present in peripheral blood samples or other body fluids are regarded as very promising, non-invasive, early cancer detection tests .
In summary, the analysis performed on two amplicons revealed that MR-SNuPE reactions performed on templates generated by conventional unbiased PCR already reach high levels of detection, that is, at least one methylated copy could easily and unambiguously be detected in a background of 1,000 unmethylated copies. However, in MR-SNuPE reactions, detection of co-methylated templates is favored because oligos are used that include CpGs (oligos 28 and 45; oligos 44 and 62). Extension primers without CpGs (oligos 27 and 30) bind independently of the methylation state (MS-SNuPE reaction) and might not be as sensitive but allow simultaneous detection of co-amplified non-methylated templates and, hence, serve as a good HM-PCR (or MS-PCR) quality control (see below).
To enhance the sensitivity we then combined SNuPE reactions on PCR products produced by HM-PCR. To define the limit of detection in such double selective reactions for very low DNA concentrations such as in blood plasma DNA samples, we again performed a proof of principle experiment. We spiked defined amounts of methylated templates (6.25, 12.5, 25, 50 and 100 pg) into unmethylated background (50 ng) and performed primer extension reactions on 10 to 24 HM-PCR reactions for each 'dilution'. For SEPT9 we observed a 100% detection down to 25 pg of spiked methylated template (corresponding to 1 methylated copy in 500 to 2,000 unmethylated copies) (see Additional file 1, supplemental Figure 2A, supplemental Figure 2B and supplemental table 1). A detection rate of 83.3% was obtained with 12.5 pg (1 in 4,000) and 58.3% with 6.25 pg (1 in 8,000) methylated template. Hence, with a 90% probability (Probit analysis), the method allows detection of 13.6 pg (CI 9.6 to 193 pg) of methylated copies in an excess of unmethylated background for SEPT9 and 21.9 pg (CI 13.0 to 36.9 pg) for TMEFF2 (Additional file 1, supplemental Figure 2C, supplemental Figure 2D). Hence, the combined HM-PCR/MR-SNuPE assay reaches sensitivities close to the theoretical optimum.
By combining two highly selective DNA methylation enrichment techniques (HM-PCR and MR-SNuPE), we generated a novel assay with extremely sensitive detection rates. With this combination, we were able to detect 14 pg methylated template DNA (~ four to five haploid copies) in 50 ng of unmethylated DNA. The method reached a detection level close to the theoretical limit. We then performed a small pilot study on plasma DNA samples from 20 patients with CRC and 20 healthy individuals to demonstrate that the method can in principle be used for routine analysis in biomedical research.
All of the main assays such MS-PCR, MethyLight or HM-PCR used for the detection of rare methylation in DNA samples are based on selective bisulfite-PCR amplification, but differ in the manner of data readout and the level of sensitivity [20–23]. In comparison to the real-time-based methods, MethylLight and other HM-PCR-based assays, the combined HM-PCR/MR-SNuPE is a simple end-point analysis that is comparable with MS-PCR but is more sensitive and selective. It does not require sophisticated chemistry but still achieves an excellent detection level. Moreover, by using combinations of MS-SNuPE and MR-SNuPE primers, it is possible to assess several CpG positions independently and also to quality control the selective methylation-specific PCR performance (see below). Very recently, another alternative assay reaching a similar sensitivity was reported. That method uses heat-stable restriction enzymes during PCR . By contrast, our method is not limited to the presence of distinct restriction sites in the amplicon of interest and, hence, is more flexible in its application. A further advantage is that it does not require extensive purification steps or complex and costly nucleotide chemistry for the analysis.
In our experience, the combination of SNuPE assays with HM-PCR assays (and presumably MS-PCR also) requires very little HPLC optimization. However, great care should be taken with primer design for both MS-SNuPE and MR-SNuPE reactions. We recommend using 12-16 nucleotides long HPLC-purified oligos. To achieve the highest selectivity and sensitivity in MR-SNuPE reactions, the oligos should encompass at least three CpG dinucleotides. In addition, any complementarity with the blocker used in HM-PCR should be avoided. For optimum HPLC separation, primers should be generated from the A-rich strand to generate either ddCTP or ddTTP extensions. Although the number of methylation-specific CpG positions in the extension oligos (such as oligo 45 that includes four CpGs) enhances the specificity for methylated products only, the detection sensitivity is equally good with oligos that additionally detect co-amplified non-methylated templates in the HM-PCR reaction (see oligo 27 in Figure 2B (g), for example). We recommend including at least one unbiased (MS-SNuPE) extension primer (oligos 27 and 30 in our example) in the assay. This allows simultaneous detection of unmethylated and methylated products, serves as a direct quality control for the HM-PCR (MS-PCR) amplification, and monitors the accuracy of the SNuPE extension. Besides the high sensitivity and specificity, the combination of HM-PCR and SNuPE detection has several practical advantages for routine sensitive diagnostics. First, the inclusion of a blocker for unmethylated DNA into the HM-PCR reaction significantly reduces the amount of unmethylated byproducts and enhances the sensitivity and specificity of the SNuPE detection. A similar principle is used in MS-PCR assays when primers are placed across several methylated CpG positions . In contrast to MS-PCR, the use of unbiased primers and the presence of selective blockers in our assay reduce but do not eliminate the amplification of unmethylated DNA. Moreover, the assay does not require complete co-methylation of all CpG positions. Secondly, our approach represents an all-or-nothing detection, that is, methylated DNA templates (above the detection threshold) always provide strong signals/peaks so that misinterpretations (false positives) can almost invariably be excluded.
Because the detection limit may easily be reached in plasma DNA samples (as seen in our small study) and because individual DNA samples might stochastically vary in the amount of tumor DNA present, the problem of false negatives has to be considered. We therefore suggest performing reactions in triplicate and, if possible, on independent DNA isolates and on several genes/amplicons. We are convinced that this robust combined HM-PCR/MR-SNuPE assay will be a useful diagnostic tool for the fast and cost-efficient detection of rare DNA methylation and will be applicable to larger cohort studies.
The described HM-PCR/MR-SNuPE assay is a robust and versatile endpoint analysis method for high sensitive detection of rare DNA methylation in complex DNA samples. The assay is cost-efficient and allows semi-automated processing of samples, making it applicable for routine testing in DNA samples isolated from biopsy tissues or body fluids.
DNA and sample material
Universal in vitro methylated human DNA was obtained from Millipore. Unmethylated DNA was prepared by molecular displacement amplification (MDA) of 10 ng peripheral blood leukocyte DNA (Promega, Mannheim, Germany) using a commercial kit (Repli-G Kit; Qiagen, Hilden, Germany). Blood plasma from 20 patients with CRC (stages I, II, III) and 20 colonoscopy-verified normal controls was obtained from Oncomatrix and ProteoGenex, where samples were collected after informed consent and in compliance with local guidelines.
Oligos used in the assays
HM-PCR, primer extension and HPLC separation
For methylation analysis by SNuPE, HM-PCR was performed as described above, omitting the probe. An aliquot (5 μl) of the HM-PCR product were treated with 1 μl of Exo-SAP (1:10 mixture of exonuclease I and SAP (USB, Staufen, Germany) for 30 min at 37°C. To inactivate the Exo-SAP enzymes, the reaction was incubated for 15 minutes at 80°C. Primer extension was carried out as described previously.
For SEPT9, to the PCR product/Exo-SAP mix, 2 μl of 10× buffer C (Solis BioDyne), 2.4 μl of 30 μM SNuPE primer (oligos 27, 28 and 45), 1 μl of 1 mM ddCTP and ddTTP or ddGTP and ddATP, respectively, and 0.5 μl of Termipol DNA polymerase (5 U/μl, Solis BioDyne) were added to reach a final volume of 20 μl. Reactions were performed in a thermal cycler under the following conditions: 96°C for 2 minutes, followed by 50 cycles 96°C for 30 seconds, 50°C for 30 seconds and 60°C for 2 minutes. Separation of SNuPE products was conducted at 50°C by continuously mixing buffer B (0.1 M triethylammonium acetate (TEAA), 25% acetonitrile) to buffer A (0.1 M TEAA) over 10 minutes, resulting in a buffer B concentration of 28% to 35% for oligo 27, 20% to 27% for oligo 28, and 21% to 27% for oligo 45.
For TMEFF2, To the PCR product. Exo-SAP mix, 2 μl of 10× buffer C (Solis BioDyne), 2.4 μl of 30 μM SNuPE primer (oligos 30, 44 and 62), 1 μl of 1 mM ddCTP and ddTTP or ddGTP and ddATP, respectively, and 0.5 μl of Termipol (5 U/μl; Solis BioDyne) were added to reach a final volume of 20 μl. Reactions were performed in a thermal cycler under the following conditions: 96°C for 2 minutes followed by 50 cycles of 96°C for 30 seconds, 60°C for 30 seconds and 60°C for 2 minutes. Separation of SNuPE products was conducted at 50°C by continuously mixing buffer B (0.1 M TEAA, 25% acetonitril) to buffer A (0.1 M TEAA) over 10 minutes, resulting in a buffer B concentration of 21% to 29% for oligo 30, 16% to 26% for oligo 44, and 18% to 28% for oligo 62.
Cloning and bisulfite sequencing of methylated and unmethylated SEPT9 amplicons
For preparation of methylated and unmethylated SEPT9 amplicons, 5 ng of bisulfite-treated methylated and unmethylated DNA, respectively, were amplified by HM-PCR without using a probe. PCR products were cloned into pGemT vector (Promega, Mannheim, Germany) according to the manufacturer's protocol, and transformed into Escherichia coli TOP10 cells. Positive clones were checked by colony PCR and subsequently sequenced. Complete unmethylated and methylated clones were used for HPLC separation.
Limit of detection
In total, 50 ng of bisulfite-treated unmethylated DNA was spiked with subnanogram amounts (100, 50, 25, 12.5, 6.25, 0 pg) of bisulfite-treated methylated DNA and analyzed in replicate by both methods in parallel. The limit of detection was defined as the minimum amount of bisulfite-treated methylated DNA that could be distinguished from unmethylated background with 90% confidence.
We are grateful for the technical support provided by Transgenomic for the WAVE IP/RP-HPLC analyses. This work was supported by the BMBF program BioChancePlus #0313166.
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