Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 36 (7), e42

Sensitive Melting Analysis After Real Time- Methylation Specific PCR (SMART-MSP): High-Throughput and Probe-Free Quantitative DNA Methylation Detection

Affiliations

Sensitive Melting Analysis After Real Time- Methylation Specific PCR (SMART-MSP): High-Throughput and Probe-Free Quantitative DNA Methylation Detection

Lasse S Kristensen et al. Nucleic Acids Res.

Abstract

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 (p16(INK4a)) 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.

Figures

Figure 1.
Figure 1.
A schematic overview of SMART-MSP. Bisulphite-modified DNA is amplified in real time using a HRM-compatible intercalating dye to obtain quantitative data. After real-time PCR a HRM step is performed for quality control of the amplicon. The interpretation is made by considering both the real-time PCR and the HRM information. Two different types of SMART-MSP amplicon design are shown here, in combination with the melting profiles and amplification data that can be expected (vertical rows), in different methylation and conversion situations (horizontal rows). Incomplete conversion can be detected most readily when non-CpG cytosines are found in between the primers and no CpG cytosines are found. By including CpG cytosines in between the primers, it can be determined if the region is heterogeneously methylated or unmethylated. If the CpG cytosines in between the primers are unmethylated, the amplification might be a result of false priming. N and M are theoretical temperatures dependent on the amplicon size and sequence.
Figure 2.
Figure 2.
Melting profiles of a true positive result for each SMART-MSP assay. Universally methylated template was amplified and analysed by HRM analysis. Each assay has a characteristic melting profile. (A) The CDH1 assay. (B) The DAPK1 assay. (C) The CDKN2A assay. (D) The RARB assay.
Figure 3.
Figure 3.
The sensitivity of the SMART-MSP assays. In all assays, the 0.1% methylated standard could be detected with high reproducibility. (A) The CDH1 assay. (B) The DAPK1 assay. (C) The CDKN2A assay. (D) The RARB assay.
Figure 4.
Figure 4.
The quantitative accuracy of the SMART-MSP assays. The quantitative accuracy of the SMART-MSP technology was assessed using the 2(−ΔΔCT) quantification approach. For each assay the calculated gene/control ratio for each standard is plotted against the dilution factor in a double logarithmic diagram. All assays proved to be quantitatively precise. (A) The CDH1 assay. (B) The DAPK1 assay. (C) The CDKN2A assay. (D) The RARB assay.
Figure 5.
Figure 5.
Validation of the conversion control in the DAPK1 and CDKN2A assays. A peripheral blood control sample was bisulphite treated using different times of conversion (20 min, 40 min, normal protocol), and used to test the conversion control of these assays. (A) The DAPK1 assay. A gradual right-shift of the melting peaks was observed as the treatment time decreases. The observed right-shift of the incompletely treated samples indicates that some of the non-CpG cytosines in between the primers were not converted. Thus, these samples could be identified as false positives. (B) The CDKN2A assay. The 40 min treated sample and the 20 min treated sample both showed right-shifted melting peaks. Again, indicating that some of non-CpG cytosines in between the primers were not converted, and thus these samples could also be identified as false positives.
Figure 6.
Figure 6.
Detection of false priming from a whole genome amplified template. We used an assay that selected poorly against unmodified templates. In this assay, five non-CpG cytosines and no CpG sites are found in between the primers. These non-CpG cytosines were converted to uracil in the bisulphite-modified template (red), but not in the unmodified template (blue). Thus, a significant right-shift of the melting profile of the unmodified amplicon is observed. (A) Real-time PCR amplification data. (B) First derivative melting peaks.
Figure 7.
Figure 7.
Identification of false positives in the CDH1 SMART-MSP assay. The CDH1 SMART-MSP assay was performed with an additional 10 cycles to obtain amplification from the unmethylated control (WGA product) shown in green. (A) Real-time PCR amplification data. (B) The melting peak of the fully unmethylated control was left-shifted by ∼1.2°C relative to melting peaks of the standards containing methylated template, and could thus be identified as a false-positive result.
Figure 8.
Figure 8.
Screening of cell lines for CDH1 methylation and breast cancer samples for RARB methylation. (A) CDH1 SMART-MSP amplification data for the positive cell lines. Five out the 14 cell lines screened were shown to be methylated at the CDH1 promoter. (B) CDH1 MethyLight amplification data from the positive cell lines. The data from the MethyLight assay was consistent with the data from the SMART-MSP assay. (C) RARB SMART-MSP amplification data for the positive tumour samples. Six out the 24 samples screened were shown to be methylated at the RARB promoter. (D) RARB MethyLight amplification data from the positive tumour samples. The data from the MethyLight assay was consistent with the data from the SMART-MSP assay.

Similar articles

See all similar articles

Cited by 65 PubMed Central articles

See all "Cited by" articles

References

    1. Gardiner-Garden M, Frommer M. CpG islands in vertebrate genomes. J. Mol. Biol. 1987;196:261–282. - PubMed
    1. Esteller M. Aberrant DNA methylation as a cancer-inducing mechanism. Annu. Rev. Pharmacol. Toxicol. 2005;45:629–656. - PubMed
    1. Herman JG, Baylin SB. Gene silencing in cancer in association with promoter hypermethylation. N. Engl. J. Med. 2003;349:2042–2054. - PubMed
    1. Laird PW. The power and the promise of DNA methylation markers. Nat. Rev. Cancer. 2003;3:253–266. - PubMed
    1. Shi H, Wang MX, Caldwell CW. CpG islands: their potential as biomarkers for cancer. Expert Rev. Mol. Diagn. 2007;7:519–531. - PubMed

Publication types

Substances

Feedback