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

Institute of Human Genetics, University of Aarhus, Wilhelm Meyers Alle 240, Aarhus C, Denmark.
Expert Review of Molecular Diagnostics (Impact Factor: 4.27). 07/2010; 10(5):575-80. DOI: 10.1586/erm.10.46
Source: PubMed

ABSTRACT 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.

Download full-text


Available from: Tine Hørning Morthorst, Jun 19, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The methylation of cytosine bases in DNA commonly takes place in the human genome and its abnormality can be used as a biomarker in the diagnosis of genetic diseases. In this paper we explore the effects of cytosine methylation on the conductance of DNA. Although the methyl group is a small chemical modification, and has a van der Waals radius of only 2 Å, its presence significantly changes the duplex stability, and as such may also affect the conductance properties of DNA. To determine if charge transport through the DNA stack is sensitive to this important biological modification we perform multiple conductance measurements on a methylated DNA molecule with an alternating G:C sequence and its non-methylated counterpart. From these studies we find a measurable difference in the conductance between the two types of molecules, and demonstrate that this difference is statistically significant. The conductance values of these molecules are also compared with a similar sequence that has been previously studied to help elucidate the charge transport mechanisms involved in direct DNA conductance measurements.
    Journal of Physics Condensed Matter 04/2012; 24(16):164204. DOI:10.1088/0953-8984/24/16/164204 · 2.22 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The terms methylation quantification and qualification seem self-explanatory however, the results of experiments aiming to quantify or qualify locus specific methylation in clinical material are often difficult to interpret. There are three main reasons for difficulties in understanding methylation status measurement. First, the complexity of locus specific methylation patterns, which oscillate between unmethylated, fully methylated, and heterogeneously methylated. Second the interpretation of methylation-screening results can frequently be problematic due to limitations of the methods used. And finally the specifications of the clinical samples used in laboratory practice frequently hamper the methylation measurement. Thus, the process of quantification and qualification of methylation has to be discussed with consideration of the specific locus analyzed, the methodology used, and the clinical material source used in each specific experiment. The question of the clinical significance of determination of different methylation levels is even more complicated, with substantial evidence for correlation between qualitative methylation changes and clinical features of the disease and at the same time no data showing that different relative levels of methylation alter the disease outcome. The limitations of methylation quantification and qualification are discussed in this mini-review.
    Frontiers in Genetics 02/2012; 3:21. DOI:10.3389/fgene.2012.00021
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Recently it has been suggested that acquisition of methylation of the BRCA1 promoter detectable in peripheral blood (PB) DNA, could give raise to development of breast cancer. In this study, we aimed to investigate a relationship between methylation of three breast cancer related genes in PB DNA, and tumor specific (somatic) methylation of these genes in the same individual. We have examined methylation status of the BRCA1, APC and RASSF1A promoter regions in a panel of 75 breast tumor and PB DNA samples from the same individual. In our study group, 4.0% of the patients displayed methylation of BRCA1 and APC in both tumor and the corresponding PB DNA. At the same time despite of marked methylation in tumor DNA, no methylation of BRCA1 and APC was seen in PB DNA of 4.3% and 2.7% of the patients respectively. The RASSF1A promoter did not show methylation in PB DNA. Our results show that for at least a subset of cancer patients methylation of certain cancer related genes in PB DNA does not seem to be directly linked to somatic methylation of the same genes in tumor DNA, and therefore may only be specific to PB DNA.
    Diagnostic Pathology 11/2011; 6:116. DOI:10.1186/1746-1596-6-116 · 2.41 Impact Factor