Methylation‐sensitive, single‐strand conformation analysis (MS‐SSCA): A rapid method to screen for and analyze methylation

  title={Methylation‐sensitive, single‐strand conformation analysis (MS‐SSCA): A rapid method to screen for and analyze methylation},
  author={Tina Bianco and Damian James Hussey and Alexander Dobrovic},
  journal={Human Mutation},
We have developed methylation‐sensitive, single‐strand conformation analysis (MS‐SSCA) as a method of screening for methylation changes. Bisulfite modification converts cytosines to thymines, but methylated cytosines remain unchanged. This modification creates sequence differences between methylated and unmethylated samples, which can be resolved by SSCA. SSCA is 70–95% efficient at detecting single base changes in a fragment. As bisulfite modification of methylated DNA would typically involve… 

Promoter Methylation Analysis on Micro-dissected Paraffin-Embedded Tissues Using Bisulfite Treatment and PCR-SSCP

Methylation-sensitive single-strand conformation analysis (MS-SSCA) is a new method of screening for DNA methylation changes that allows the detection of methylation of the p16 gene promoter and is successful in analyzing almost all tumor samples microdissected from archival paraffin-embedded fixed tissue sections and obtaining reproducible results.

A methylation sensitive dot blot assay (MS-DBA) for the quantitative analysis of DNA methylation in clinical samples

A methylation sensitive dot blot assay (MS-DBA), which is quantitative and combines bisulfite modification, PCR amplification using primers without CpG sites, and dot blot analysis with two probes specific for methylated and unmethylated DNA, can facilitate the routine analysis of DNA methylation in all types of clinical samples.

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes.

DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

DNA methylation: a profile of methods and applications.

A review of methods designed to yield quantitative and qualitative information on genomic DNA methylation hopes to be a valuable tool in selecting the best techniques to address particular questions concerning the cytosine methylation status of genomic DNA.

DNA Methylation: A Timeline of Methods and Applications

The major developments in the methodologies used over the past three decades to examine the elusive epigenome (or methylome) are described, with molecular techniques being employed to indirectly examine DNA methylation levels at both a genome-wide and locus-specific context.

Methylation-sensitive high-resolution melting

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 and enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.

A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples

It is shown that for the model system, RARB2 P2 promoter, use of MRED increased informative sequencings ninefold, and MRED did not alter the clonal representation in one fully methylated cell line, H-596, treated or not with 5-azadeoxycytidine, a methylation inhibitor.

DNA methylation: Bisulphite modification and analysis

The 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution is described.



Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

The use of MSP is demonstrated to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin and von Hippel-Lindau) in human cancer.

Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE).

This methylation-sensitive technique has several advantages over existing methods used for detection of methylation changes because small amounts of DNA can be analyzed including microdissected pathology sections and it avoids utilization of restriction enzymes for determining the methylation status at CpG sites.

High sensitivity mapping of methylated cytosines.

A genomic sequencing technique which is capable of detecting every methylated cytosine on both strands of any target sequence, using DNA isolated from fewer than 100 cells is developed.

A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.

A genomic sequencing method is reported that provides positive identification of 5-methylcytosine residues and yields strand-specific sequences of individual molecules in genomic DNA, which suggests that the high methylation level of single-copy sequences in sperm may be locally modulated by binding of protein factors in germ-line cells.

CpG methylation within the 5′ regulatory region of the BRCA1 gene is tumor specific and includes a putative CREB binding site

The first evidence suggesting a role for DNA methylation in the transcriptional inactivation of the BRCA1 in human breast cancer is presented, with screening of randomly sampled breast carcinomas revealed the presence of CpG methylation adjacent to the B RCA1 transcription start site.

Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia.

Evidence is presented that the higher frequency of p15INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p 15inks4B-methylated lymphocytes in normal blood.

The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions.

The results illustrate the need to keep the size of the PCR fragment small when performing SSCP to detect mutations, and the position of the base substitution was more important than the precise base substitution in determining whether a mutation was detected.

Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors.

The dynamics of DNA methylation in cancer cells are clearly different from normal cells and an insight into the mechanism of abnormal methylation of CpG islands incancer cells is given.

COBRA: a sensitive and quantitative DNA methylation assay.

COBRA shows that methylation levels in the original DNA sample are represented by the relative amounts of digested and undigested PCR product in a linearly quantitative fashion across a wide spectrum of DNAmethylation levels.