Which DNA fragment as control for complete bisulfite conversion - (Oct/11/2005 )
According to my recent results, it looks like there are several imcomplete treatment the PCR fragment. So I hope there would be an internal control to show the bisulfit treatment.
Based on my observation, fragments with dense CpGs tend to be refractory to bisulfite conversion. I think such fragments such as the Ecadherin CpG island would be a good control.
evidence of incomplete bisulfite treatment could also arise from ineffective primer design. You may want to pick different primers for amplifying your region of interest.
I also believe that the different stratge for primer design would remove the incomplete bisulfit sequence. For example, I think amplifying a short fragment with the converted sequence speicific primer. I'm a beginner and I like to hear more infomation about primer design.
I'm very glad to know that Ecadherin CpG can be used as a control. Can you mail the reference about region. (my mail address is firstname.lastname@example.org) Thank you very much.
The following is the Ecadherin promoter with the CpG island in red. But I don't think you really need to amplify another region unrelevant to your research as control. From looking at the conversion of non-cpg C in the region of your interest, you will know if there is incomplete conversion.
1 tctagaaaaa ttttttaaaa aattaggccg ctcgagcgag agtgcagtgg
51 ctcacgcctg taatccaaca cttcaggagg ctgaagaggg tggatcacct
101 gaggtcagga gttccagacc agcctggcca acatggtgaa accccgtctt
151 gtactaaaaa tacaaaatta gccggtgtgg tggcacacgc ctgtagtccc
201 agctactcaa taggctgaga caggagagtc tcttgaaccc ggcaggcgga
251 ggttgcagtg agccgagatc gtgccactgc actccagcct gggcaagaca
301 gagcgagact ccgtctcaaa aaatacaaac aaaacaaaca aacaaaaaat
351 taggctgcta gctcagtggc tcatggctca cacctgaaat cctagcactt
401 tgggaggcca aggcaggagg atcgcttcag cccaggagtt cgagaccagg
451 ctgggcaata cagggagaca cagcgccccc actgcccctg tccgccccga
501 cttgtctctc tacaaaaagg caaaagaaaa aaaaaattag cctggcgtgg
551 tggtgtgcac ctgtactccc agctactaga gaggctgggg ccagaggacc
601 gcttgagccc aggagttcga ggctgcagtg agctgtgatc gcaccactgc
651 actccagctt gggtgaaaga gtgagcccca tctccaaaac gaacaaacaa
701 aaaatcccaa aaaacaaaag aactcagcca agtgtaaaag ccctttctga
751 tcccaggtct tagtgagcca ccggcggggc tgggattcga acccagtgga
801 atcagaaccg tgcaggtccc ataacccacc tagaccctag caactccagg
851 ctagagggtc accgcgtcta tgcgaggccg ggtgggcggg ccgtcagctc
901 cgccctgggg aggggtccgc gctgctgatt ggctgtggcc ggcaggtgaa
951 ccctcagcca atcagcggta cggggggcgg tgctccgggg ctcacctggc
1001 tgcagccacg caccccctct cagtggcgtc ggaactgcaa agcacctgtg
1051 agcttgcgga agtcagttca gactccagcc cgctccagcc cggcccgacc
1101 cgaccgcacc cggcgcctgc cctcgctcgg cgtccccggc cagccatggg
1151 cccttggagc cgcagcctct cggcgctgct gctgctgctg cag
check out this topic Bisulfite Primer Design
and do read up on the paper listed there, it has all the parameters for designing Primers.
Thank you PCRman and Nick.
I want to amplify the fragment in a longer region, but worried it will introduce some other bias, say, a short target region with BSP speicific primer will ensure the treatment of the target region.
So my question is based on the good quality of genomic DNA and standard treatment, is it possible there still remains some unconverted nucleotides? Has anybody test this case with the non-BSP specific primer in a relative longer region(400bp)?
Another question is it seems E-cadherin expresses in many different cell types, but there still lacks the information whehter this gene express in the myocyte and neurons?