How to reduce the bisulfite PCR bias? - (Jun/18/2010 )
Hi,
By using bisulfite treated DNA I was able to amplifed several genes. What I did is first to convert DNA with Qiagen epiTech Kit. Then use Taq to amplify the target genes. So far the results for unmethylated genes look pretty OK as on C peak can be detected from the histogram. However, for those methylated segments (detected by restriction enzyme PCR), the results vary in a high degree. For one part of DNA, the plus strand has a high methylation rate whereas the minus strand can barely see any methylation. By checking literatures I noticed that this discrenpancy could be resulted from the preference of amplification to either unmethylated or methylated strands. Here I am wondering if anyone has experience with this matter and any precious advices are most welcome.
Hi Laver,
I am a little confused. You said "the plus strand has a high methylation rate whereas the minus strand can barely see any methylation". Do you mean you amplified both the sense strand and the antisense strand, and then sequence both strands? or you amplified the sequence strand, and then sequence the product from both ends?
I mean design primers for plus and minus strand seperately and then amplified and sequence the PCR products.
pcrman on Jun 28 2010, 12:30 AM said:
I am a little confused. You said "the plus strand has a high methylation rate whereas the minus strand can barely see any methylation". Do you mean you amplified both the sense strand and the antisense strand, and then sequence both strands? or you amplified the sequence strand, and then sequence the product from both ends?
I mean design primers for plus and minus strand seperately and then amplified and sequence the PCR products.
pcrman on Jun 28 2010, 12:30 AM said:
I am a little confused. You said "the plus strand has a high methylation rate whereas the minus strand can barely see any methylation". Do you mean you amplified both the sense strand and the antisense strand, and then sequence both strands? or you amplified the sequence strand, and then sequence the product from both ends?
One way to reduce your PCR bias is to eliminate amplification all together using a PCR Free or Amplification Free library preparation method.
Getting rid of PCR will give you better read mapping, a reduction in duplicate sequences and much better representative base identities. The kit is called the NEXTflex PCR-Free Sequencing Kit.
good luck with your sequencing.
Laver on Tue Jun 29 20:50:13 2010 said:
I mean design primers for plus and minus strand seperately and then amplified and sequence the PCR products.
Hi Laver,
I am a little confused. You said "the plus strand has a high methylation rate whereas the minus strand can barely see any methylation". Do you mean you amplified both the sense strand and the antisense strand, and then sequence both strands? or you amplified the sequence strand, and then sequence the product from both ends?