What is the best way to analyze methylation in bisulfite treated DNA?
The first reported method of methylation analysis using bisulfite-treated DNA utilized PCR and standard dideoxynucleotide DNA sequencing to directly determine the nucleotides resistant to bisulfite conversion.
Does bisulfite remove 5 methylcytosine?
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 is the recovery of the bisulfite-converted DNA determined?
When determining the recovery of the bisulfite-converted DNA, two primary factors need to be considered 1.) The intactness of the starting material, and 2.) RNA contamination. The quality of the DNA, in terms of size, used as input for bisulfite conversion is the most important factor when assessing recovery.
What is the role of bisulfite in the cytosine region?
In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity . Treatment of DNA with bisulfite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected.
Which nucleotide base is methylated at the 5 position?
CytosineDNA (de)methylation dynamics. Cytosine (C) is methylated at 5′ carbon of the pyrimidine ring by DNA methyltransferases (DNMT-1, DNMT-3A, DNMT-3B), the writer, to generate 5-methylcytosine (5-mC), which is recognized by methyl-CpG binding protein 2 (MeCP2), the reader.
At what site does DNA become methylated?
cytosine basesToday, researchers know that DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5-methylcytosine by DNA methyltransferase (DNMT) enzymes.
How is DNA methylation status is determined using bisulfite treatment?
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. DNA methylation status can be determined by direct PCR sequencing or cloning sequencing.
What is bisulfite methylation?
Bisulfite Conversion is a process in which genomic DNA is denatured (made single-stranded) and treated with sodium bisulfite, leading to deamination of unmethylated cytosines into uracils, while methylated cytosines (both 5-methylcytosine and 5-hydroxymethylcytosine) remain unchanged.
What happens when DNA is methylated?
DNA methylation, a process of adding a methyl group to DNA done by a DNA methyltransferase is a heritable (epigenetic) alteration leading to cancer, atherosclerosis, nervous disorders (Imprinting disorders), and cardiovascular diseases.
How is DNA methylation used in DNA repair?
DNA methylation status is highly polymorphic and can be reshaped during and after DNA damage-repair events. Over time, the DNA methylation profiles of Rec H and Rec L cells stabilize and generate cells with different but heritable GFP expression levels.
How does methylation sequencing work?
Most methods rely on bisulfite conversion of DNA to detect unmethylated cytosines. Bisulfite conversion changes unmethylated cytosines to uracil during library preparation. Converted bases are identified (after PCR) as thymine in the sequencing data, and read counts are used to determine the % methylated cytosines.
How does methylation PCR work?
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 is bisulfite sequencing used for?
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 perform bisulfite sequencing?
The five basic steps in bisulfite conversion are: 1) DNA denaturation; 2) incubation with bisulfite at elevated temperature; 3) removal of bisulfite by desalting; 4) desulfonation of sulfonyl uracil adducts at alkaline pH; and 5) removal of the desulfonation solution.
Which of the following can be measured using bisulfite conversion and then sequencing?
Bisulfite sequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied.
How long is bisulfite sequencing?
Following bisulfite treatment and purification, PCR amplification along with PCR product purification requires about 3 hours. Samples are then prepared for sequencing. If sending the samples to a sequencing core, time requirements may vary, but 3 days should be allotted for sequencing by a core facility.
What are the two factors that determine the recovery of bisulfite-converted DNA?
When determining the recovery of the bisulfite-converted DNA, two primary factors need to be considered 1.) The intactness of the starting material, and 2.) RNA contamination. The quality of the DNA, in terms of size, used as input for bisulfite conversion is the most important factor when assessing recovery.
What is the best way to analyze bisulfite-converted DNA?
Bisulfite sequencing is still among the most common techniques used for analyzing bisulfite-converted DNA and provides single base resolution across the entire amplicon. Cloning followed by sequencing with vector-specific primers is recommended to obtain the best sequencing results for quantification of methylation.
What is methyl specific PCR?
Methylation Specific PCR (MSP) relies on amplification to assess the methylation status at specific CpG sites. Success with this system depends on the differential amplification of the template using methylated (M) and non-methylated (U) primer sets. While most of the considerations for primer design are identical to those for bisulfite PCR, treatment of CpG sites within the primer is completely different. For MSP it is necessary to locate CpG sites at the 3'-end of the primers with cytosines in the methylated (M) primers and thymines in the non-methylated (U) primers.
How long should a bisulfite primer be?
Primer design is the key to successful bisulfite PCR. Unlike normal PCR, bisulfite PCR primers need to be long (usually between 26-30 bases ) and the amplicon size should be relatively short ...
What happens when you use bisulfite?
Treatment with bisulfite is inherently a harsh process that dramatically changes both the chemical makeup and physical properties of the DNA. Input DNA transforms from a large, stable, double-stranded molecule to a collection of randomly fragmented, single-stranded fragments having almost all cytosines completely changed to uracil.
How long to chill ethidium bromide gel?
Chilling the gel for several minutes in an ice bath will force enough base-pairing to allow intercalation of the ethidium bromide for the DNA to be visible. The converted DNA will run as a smear, generally from > 1,500 down to 100 bp.
Where should CPG be located on primers?
Ideally, the primers should not contain CpG sites, however, if they are necessary locate them at the 5'-end of the primer with a mixed base at the cytosine position. It is also important to note that only one strand of the bisulfite-converted template will be amplified by any given primer set.