The bisulfite conversion can be checked using Methylation Specific-PCR or MS-PCR, Bisulfite sequencing and also Methylation based microarrays.
Full Answer
What are the considerations when starting with bisulfite conversion?
When starting with bisulfite conversion, there are a number of considerations to keep in mind to ensure optimal performance and subsequent accuracy of the DNA methylation analysis. Treatment with bisulfite is inherently a harsh process that dramatically changes both the chemical makeup and physical properties of the DNA.
What is the bisulfite modification technique?
The bisulfite modification technique uses bisulfite salt to deaminate cytosine residues on single-stranded DNA, converting them to uracil while leaving 5-methylcytosine unchanged (Figure 1). Figure 1. Example sequence of an unmethylated or methylated DNA template after bisulfite conversion. mC = methylated cytosine.
What is bisulfite conversion of DNA?
Sodium bisulfite conversion of DNA is the fastest, easiest, and most popular way to study DNA methylation analysis and selecting the right bisulfite conversion kit can be the difference between accurate data and frustrating wasted hours spent in the lab.
What is the incubation temperature for bisulfite PCR?
Replicate bisulfite PCRs were incubated in a gradient thermal cycler using an annealing temperature gradient of 55 to 65oC (left to right). Equal volumes from each reaction were analyzed in a 2% agarose gel with a 100 bp DNA marker.
How do you measure bisulfite conversion?
Hi, The bisulfite conversion can be checked using Methylation Specific-PCR or MS-PCR, Bisulfite sequencing and also Methylation based microarrays.
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.
What is the purpose of bisulfite conversion?
Bisulfite Conversion is a method used to make it easy to distinguish methylated from unmethylated cytosines in genomic DNA at single base resolution. DNA is first denatured (made single-stranded) and then treated with sodium bisulfite.
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.
How much DNA is needed for bisulfite sequencing?
Commonly 10–20 samples are sufficient, but for some applications as many as 50 may be necessary.
What is Cobra analysis?
Combined Bisulfite Restriction Analysis (or COBRA) is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA.
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.
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 is long read bisulfite sequencing?
Detection of base modifications by bisulfite and long-read sequencing. Whole-genome bisulfite sequencing (WGBS) provides accurate binary calls of cytosine methylation status at nucleotide resolution, but cannot distinguish between 5mC and 5hmC or detect other oxidised forms without additional techniques.
What type of sequencing must be used to detect DNA methylation?
3.1. Bisulfite Sequencing. The technique of bisulfite sequencing is considered to be the “gold standard” method in DNA methylation studies. Current DNA sequencing technologies do not possess the ability to distinguish methylcytosine from cytosine.
How do Illumina methylation arrays work?
The Illumina Methylation Assay using the Infinium I platform uses 'BeadChip' technology to generate a comprehensive genome-wide profiling of human DNA methylation. Similar to bisulfite sequencing and pyrosequencing, this method quantifies methylation levels at various loci within the genome.
How does whole genome bisulfite sequencing work?
The bisulfite-treated DNA is purified on a spin column and is used to prepare the sequencing library using the EpiGnome™ Kit (Epicentre). In this procedure, bisulfite-treated single-stranded DNA is random- primed using a polymerase able to read uracil nucleotides, to synthesize DNA containing a specific sequence tag.
Most recent answer
Dear Jody Ye ¿Could you please share the solution you found for your problem? I am facing a very similar one right now. Thanks indeed in advance
Popular Answers (1)
The first link is a PLoS paper in which different bisulfite conversion kits are compared, not only concerning conversion efficiency, but also many other factors.
All Answers (11)
As far as I know, there is no simple, quick way to check bisulfite conversion. But bisulfite sequencing using automated ABI or similar platforms could give better and reproducible results compared to manual bisulfite sequencing.
Similar questions and discussions
How is the best way for the check the bisulfite conversion and how can I measure the efficiency?
Most recent answer
Alvaro Fernando Nieto, Just beware for a negative control, most cell lines are immortalised and have atypical methylation profiles so may not be a great negative control.
Popular Answers (1)
I usually test the conversion through PCR. I use a Zymo Research kit. Basically there is a converted pattern and another does not, it uses the same primers and it checks whether or not the sample is converted.
All Answers (4)
I usually test the conversion through PCR. I use a Zymo Research kit. Basically there is a converted pattern and another does not, it uses the same primers and it checks whether or not the sample is converted.
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.
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 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.
How many cycles of amplification are needed for a reverse primer?
Only the reverse primer will actually bind to the target DNA, which in turn will generate template for the forward primer to anneal. Usually, 35 to 40 cycles are required for successful amplification.
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.
What is bisulfite conversion?
DNA bisulfite conversion is a unique tool used to discriminate between methylated and unmethylated cytosines for DNA methylation studies. Only bisulfite modification of DNA followed by sequencing yields reliable information on the methylation states of individual cytosines at single base resolution. The bisulfite modification technique uses bisulfite salt to deaminate cytosine residues on single-stranded DNA, converting them to uracil while leaving 5-methylcytosine unchanged (Figure 1).
How to measure bisulfite?
For quantitative analysis, the amount of bisulfite DNA may be measured using a fluorescence detection method such as that used for RNA – since the DNA is now single-stranded and the majority of cytosines have been converted to uracil. Measure your converted DNA relative to a blank or RNA standard to estimate your yield.
What is the DNA 260/280 ratio?
Check that your input DNA is not degraded by running a gel. The DNA 260/280 ratio should be 1.6-1.9.
Does bisulfite fragment DNA?
As mentioned above, the bisulfite process itself will fragment your DNA due to acidic ...