I learned how to perform a bisulfite conversion of DNA for methylation analysis. DNA methylation is used as an epigenetics tool to control gene expression, meaning where and when this gene should be expressed by adding a methyl group to the cytosine rings in DNA.
The purpose of a bisulfite conversion is that this process takes places after the DNA has been methylated and converts the remaining, unmethylated cytosine bases into uracil and this changes the overall DNA sequence which helps in yielding results based on a single nucleotide and reduce the differentiation between single nucleotide polymorphisms (SNP, variations of a single nucleotide, in this case cytosine and guanine)
Protocol:
- Add 130 microlitres of lightning conversion reagent to a 20 microlitres sample of DNA and centrifuge.
- Place the PCR tube in a thermal cycler under the following conditions:
98°C for 8 minutes
54 °C for 60 minutes
4 °C for storage up to 20 hours
- Add a binding buffer and centrifuge
- Add a wash buffer and centrifuge.
- Add a desulphonation buffer and incubate at room temperature for 15 minutes.
- Measure the concentration of the sample using a nano-vue spectrometer.
- Perform PCR
The purpose of a bisulfite conversion is that this process takes places after the DNA has been methylated and converts the remaining, unmethylated cytosine bases into uracil and this changes the overall DNA sequence which helps in yielding results based on a single nucleotide and reduce the differentiation between single nucleotide polymorphisms (SNP, variations of a single nucleotide, in this case cytosine and guanine)
Protocol:
- Add 130 microlitres of lightning conversion reagent to a 20 microlitres sample of DNA and centrifuge.
- Place the PCR tube in a thermal cycler under the following conditions:
98°C for 8 minutes
54 °C for 60 minutes
4 °C for storage up to 20 hours
- Add a binding buffer and centrifuge
- Add a wash buffer and centrifuge.
- Add a desulphonation buffer and incubate at room temperature for 15 minutes.
- Measure the concentration of the sample using a nano-vue spectrometer.
- Perform PCR
What is the purpose of using all those buffers? I assume it's to purify the DNA, but why do you need to use three buffers instead of just one?
ReplyDeleteThe binding buffer contains acetate, so that makes it easy to dissolve the proteins and other molecules inside the sample. The purpose of a wash buffer is to wash away any other molecules that were not dissolved by the binding buffer and the desulphonation buffer removes the sulfate group that's on the uracil when converted from cytosine.
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