What does bisulfite do to DNA?
Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Therefore, DNA that has been treated with bisulfite retains only methylated cytosines.
How bisulfite treatment coupled with PCR enable the identification of DNA methylation?
Bisulfite conversion is regarded as the gold standard in defining DNA methylation status. Treatment of DNA with sodium bisulfite converts unmethylated cytosine to uracil, which is subsequently converted to thymine during PCR amplification, while methylcytosine remains unchanged (Fig.
Is bisulfite-converted DNA single stranded?
Uracil is then converted to thymine upon PCR amplification. Note that bisulfite-converted DNA is a single-stranded DNA because the two strands of DNA are no longer complementary after conversion.
What is the basic principle of bisulfite sequencing?
Principles of methylation analysis using bisulfite genomic sequencing. After treatment with sodium bisulfite, unmethylated cytosine residues are converted to uracil whereas 5-methylcytosine (5mC) remains unaffected. After PCR amplification, uracil residues are converted to thymine.
How do you confirm bisulfite conversion?
Following bisulfite conversion the unmethylated cytosines are converted into uracil while the methylated cytosines remain as cytosine. The methylation profile of the DNA can then be determined by PCR amplification followed by DNA sequencing, where the uracils will be detected as thymine.
How much DNA is needed for bisulfite sequencing?
Up to 2 µg genomic DNA (or 1 ng purified fragment) may be used for BGS. Although 90% or more of DNA is lost during bisulfite treatment, using too much DNA may lead to incomplete deamination, due to re-annealing of complementary sequences.
What is the principle of Methylation-Specific PCR?
Methylation-specific PCR (MS-PCR or MSP) is one of the most commonly used methods for gene/sequence-specific detection of DNA methylation. The DNA undergoes bisulfite conversion of cytosine to uracil and then the methylated sequences are selectively amplified with primers specific for methylation.
What does bisulfite sequencing tell you?
Bisulfite sequencing is mainly used to detect DNA methylation patterns. As DNA methylation patterns are erased during PCR (polymerase chain reaction) amplification, current sequencing, and microarray technologies cannot distinguish between methylated and unmethylated cytosines.
How do you test for DNA methylation?
Currently, there are three primary methods to identify and quantify DNA methylation. These are: sodium bisulfite conversion and sequencing, differential enzymatic cleavage of DNA, and affinity capture of methylated DNA (1). Restriction enzyme based differential cleavage of methylated DNA is locus-specific.
Who discovered bisulfite sequencing?
The bisulfite-mediated specific deamination of cytosine was discovered in 1970 by Shapiro and coworkers in New York City and by my research group in Tokyo independently. The use of this chemical treatment for analyzing 5-methylcytosine in DNA was initiated by a group of Australian scientists in 1992.
Does methylation affect PCR?
Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR.
How does bisulfite treatment affect the methylation of DNA?
Thus, bisulfite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single-nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information.
Are there any alternative methods to sequencing bisulfite?
Alternative methods to bisulfite sequencing include Combined Bisulphite Restriction Analysis and methylated DNA immunoprecipitation (MeDIP). Methodologies to analyze bisulfite-treated DNA are continuously being developed. To summarize these rapidly evolving methodologies, numerous review articles have been written.
Which is the best bisulfite conversion kit for DNA?
The kits studied showed different but comparable results regarding DNA degradation, conversion efficiency and conversion specificity. However, the best performance was observed with the MethylEdge Bisulfite Conversion System (Promega) followed by the Premium Bisulfite kit (Diagenode).
How are microarrays used to analyze bisulfite treated DNA?
Microarray -based methods are a logical extension of the technologies available to analyze bisulfite-treated DNA to allow for genome-wide analysis of methylation. Oligonucleotide microarrays are designed using pairs of oligonucleotide hybridization probes targeting CpG sites of interest.