Bisulfite: PCR condition or primer problem? - (May/03/2007 )

you're absolutely right, it's a huge pain to find impeccable primers with MPE!
But it does a nice job of generating colorful outputs with modified bases highlighted. I'll take it manually from there.

As for the amplicon size, I believe 500bp is already quite the challenge for me, I'll save the kb amplicons for later

Another question, though: When designing hemi nested primers, is there a necessary minimum distance between the two primers that will be on the same side of the amplicon (outside and inside primer)? Is it ok for them to overlap?

Hi there, I'm trying to design MSP primers for the gene which I list below. There are 2 CpG islands according to Methyl Primer Express. It seems that one of the CpG islands spans the promoter and the exon, and the other one is located at the genome itself. Now I have problems........which CpG island should I choose?? Isn't the genome supposed to have few CpG dinucleotides?? please help me, thank you so much...

gcttgcaccatgttggacaggctagtctcaaactcctgacctcatgtgttcttcccaccttggcctcccaaagtgctggg
atcacaggtcactgcacccagcctagagtatttaatatacattcatataaaaaagaagcactggtttcataaaacaaatgt
acattatatgagattccacttcataaaagcatgtatgtgtaccccaacactaaattggcccttttaacaaaatatcagact
ttgtcctacttaactggaaagattatctatattttataaagaagagaaataacataatagtaaggctgtgatttcctcttt
tttgcaaaaactatttttgaaaagtattactgaattgataacatatatatggagagatttaaaatactagataagggagct
tattccaatgaatttacagaaaggtaggtattttagtcaactaaaaatataaataaaaattatcaattcacttatttggat
acagcagtcatgccagagtttataaatatagtgaaatcactgaagatttttttttacccccaaaactgaaaaaaatggcag
tgtaccttgtgaattttttaaatgaaaaaagaatacgaaatgatgaaggcccttatctgatgatgacaaactagaaaactg
aaatgtaaaggggaaaatgtgagtatgtgtttccctccagcaaattaaagacagggtacatagattcaagaggttgcgctt
tgaggataaaaagctgggccctgtgcagttttccaagacaacctctgggcgccgctgtcgaccaaaaggaagtgaaggctt
ctcaattctgcaggagcgtcaggcagggtgccttcctgctttgtccggtgcactgagcacagacgctgctcctgttcaccc
tcgagcgcctaactaataagtcctaagctcaaatcacagaagtccagggtgctgccatttctttttgaaaaacatcccaga
ggaaagaagctccgcggagagttcctgaaATGCAGAGAGCCAGAGAAGCCGAAATGATGAAAAGTCAGGTACTGTTTCCCT
TCCTGCTGTCTTTGTTCTGCGGGGCCATCTCCCAGCAGATCCGATACACGATTCCAGAGGAGCTAGCCAACGGCTCACGGG
TGGGGAAACTTGCCAAGGATCTGGGGCTCAGTGTCCGGGAGTTGCCAACTCGAAAACTGCGGGTTAGTGCAGAGGATTATT
TCAACGTTAGTTTGGAGAGCGGGGATTTGTTAGTGAACGGTAGGATAGATCGAGAGAAGATTTGCGGAAGGAAACTTGAGT
GTGCACTAGAATTCGAAACGGTCGCTGAAAACCCAATGAATGTTTTCCACGTGGTTGTTGTAATCCAAGATATTAATGACA
ATGCACCACGTTTCGTTGCAAAAGGCATTGACTTAGAAATTTGTGAGTCAGCCTTACCCGGGGTAAAATTCTCTCTGGATT
CTGCTCAAGATGCAGATGTGGAAGGCAATTCACTGAAGTTATACACCATCAACCCCAATCAATACTTCTCTCTGTCAACGA
AGGAAAGTCCTGATGGAAGTAAATATCCGGTATTACTGCTGGAAAAACCTCTAGACAGGGAACATCAGAGCTCTCATCGCT
TAATCCTGACTGCCATGGATGGCGGGGACCCGCCTCTAAGCGGCACCACCCATATCTGGATCCGAGTTACGGATGCCAATG
ATAATGCTCCCGTGTTTAGCCAGGAGGTATACAGGGTTAGCCTCCAAGAAAACGTACCGTGGGGAACCTCCGTGCTGCGGG
TGATGGCCACAGACCAGGATGAGGGCATTAATGCAGAGATCACCTATGCCTTCCTCAATTCCCCAATAAGTACCAGCCTCT
TCAATCTCAATCCAAATACTGGCGACATCACAACCAATGGCACATTGGATTTTGAAGAGACAAGTAGATATGTGTTGAGTG
TGGAAGCTAAGGATGGAGGAGTACACACAGCTCACTGTAATGTTCAAATAGAAATTGTTGACGAGAATGACAATGCCCCAG
AGGTGACATTCATGTCCTTCTCTAACCAGATTCCAGAGGATTCAGACCTTGGAACTGTAATAGCCCTCATAAAAGTGCGAG
ACAAGGATTCTGGGCAAAATGGCATGGTGACATGCTATACTCAGGAAGAAGTTCCTTTCAAATTAGAATCCACCTCGAAGA
ATTATTACAAGCTGGTGATTGCTGGAGCCCTAAACCGGGAGCAGACAGCAGACTACAACGTCACAATCATAGCCACCGACA
AGGGCAAACCAGCCCTTTCCTCCAGGACAAGCATCACCCTGCACATCTCCGACATCAACGACAATGCACCTGTTTTCCATC
AGGCCTCCTATGTGGTCCACGTGTCTGAGAACAACCCACCTGGCGCCTCCATTGCACAAGTAAGCGCCTCCGACCCGGATT
TGGGACCCAACGGCAGAGTCTCCTACTCTATTCTGGCCAGTGACCTGGAGCCGCGGGAGCTGTTGTCCTACGTGTCCGTGA
GCCCGCAGAGCGGGGTGGTGTTCGCGCAGCGCGCCTTCGACCACGAGCAGCTGCGCGCCTTCGAGCTCACACTGCAGGCCA
GGGACCAGGGCTCCCCCGCGCTCAGCGCCAACGTGAGCCTGCGCGTGTTGGTGGGCGACCTCAATGACAATGCGCCACGGG
TGCTGTACCCCGCGCTGGGGCCTGATGGCTCCGCCCTCTTCGATATGGTGCCACGCGCCGCAGAGCCCGGCTACCTGGTGA
CCAAGGTGGTGGCGGTGGACGCAGACTCAGGACACAACGCTTGGCTGTCCTACCACGTGCTGCAGGCCAGCGAGCCCGGGC
TCTTCAGCCTGGGGTTGCGCACGGGTGAGGTGCGCACAGCGCGTGCCTTGGGCGACAGGGACGCGCCCGCCAGCGCCTGCT
GGTCGCTGTGCGTGATGGAGGACAGCCGCCACTCTCCGCCACCGCCACGCTGCACCTAATCTTCGCGGATAGCCTGCAAGA
GGTATTGCCAGACCTCAGCGACCGCCCTGAGCCCTCTGACCCCCAGACGGAACTGCAGTTTTACCTGGTTGTGGCCTTGGC
CTTGATCTCAGTGCTCTTTCTCCTCGCGGTGATTCTAGCGATCGCCCTGCGCCTGCGACGTTCCTCCAGCCTCGACACTGA
GGGCTGCTTTCAAACCGGTCTCTGCTCCAAGTCTGGGCCCGGGGTTCCTCCCAACCACAGCGAGGGGACTTTGCCCTATTC
CTACAATCTATGTATTGCCTCTCATTCTGCAAAGACAGAGTTTAATTCTCTCAACCTGACACCGGAAATGGCTCCCCCTCA
GGATCTGCTGTGTGATGATCCTTCTATGGTTGTATGTGCCAGTAATGAAGATCACAAAATCGCTTATGACCCTTCTTTGTC
TTCGCACGTGAGTTTCTGCAAATCTAGTTAA

@cyburn,

it is fine to have them overlap for a hemi nested strategy.

@gtwu,

the best thing to do is to look if other methylation work has been done on your gene of interest. more often than not, the CpG island 5' to transcription start site is your best bet.

just having a quick look on your gene of interest, it is very interesting! there are CpG islands for all the variants! you would have to look at each individually and marry this with expression data of the variants.

Nick

Thank you, Nick~~ Would you please help me to check if these MSP primers are ok??

GTAGATTCGATATACGATTTTAGAGGAGTTAGTTAACGGTTTACGGGTGGGGAAATTTGTTAAGGATTTGGGGTTTAGTGTTCGGGAGTTGTTAATTCGAAAATTGCGGGTTAGTGTAGAGGATTATTTTAACGTTAGTTTGGAGAGCGGGGATTTGTTAGTGAACGGTAGGATAGATCGAGAGAAG
ATTTGCGGAAGGAAATTTGAGTGTGTATTAGAATTCGAAACGGTCGTTGAAAATTTAATGAATGTTTTTTACGTGGTTGTTGTAATTTA

Heminested Outside forward:
23 b.p.
5’ TGTTAAGGATTTGGGGTTTAGTG 3’
Tm= 58.34, CG=0, C=5

Inside forward:
25 b.p.
5’ CGGGAGTTGTTAATTCGAAAATTGC 3’
Tm=65.5, CG=3, C=4

Inside reverse:
22 b.p.
5’ ACGACCGTTTCGAATTCTAATA 3’
Tm=56.6, CG=3, C=3

Inside amplicon: 152 b.p.
Outside amplicon: 177 b.p.


AGTAAGCGTTTTCGATTCGGATTTGGGATTTAACGGTAGAGTTTTTTATTTTATTTTGGTTAGTGATTTGGAGTCGCGGGAGTTGTTGTTTTACGTGTTCGTGAGTTCGTAGAGCGGGGTGGTGTTCGCGTAGCGCGTTTTCGATTACGAGTAGTTGCGCGTTTTCGAGTTTATATTGTAGGTTAGGGATTAGGG
TTTTTTCGCGTTTAGCGTTAACGTGAGTTTGCGCGTGTTGGTGGGCGATTTTAATGATAATGCGTTACGGGTGTTGTATTTCGCGTTGGGGTTTGATGGTTT
CGTTTTTTTCGATATGGTGTTACGCGTCGTAGAGTTCGGTTATTTGGTGATTAAGGTGGTGGCGGTGGACGTAGATTTAGGATATAACGTTTGGTTGTTTTATTACGTGTTGTAGGTTAGCGAGTTCGGGTTTTTTAGTT
TGGGGTTG

Outside forward:
20 b.p.
5’ TTTTGGTTAGTGATTTGGAG 3’
Tm= 50.4, CG=0, C=5

Outside reverse:
22 b.p.
5’ ACCACCTTAATCACCAAATAAC 3’
Tm= 53.36, CG=0, C=5

Inside forward:
22 b.p.
5’ CGTGTTCGTGAGTTCGTAGAGC 3’
Tm=60.1, CG=4, C=4

Inside reverse:
20 b.p.
5’ GTTAACGCTAAACGCGAAAA 3’
Tm=56.64, CG=4, C=8

Inside amplicon: 124 b.p.
Outside amplicon: 304 b.p.