About DAPK1 MSP primer design. - About MSP primer design. (Nov/20/2008 )

Hi! I'm a newer, and i want to detect the Methylation state of DAPK1.
In fact, there are lots of paper about it, but I blasted the MSP primer, which had been used in published paper,
the result showed no matched gene. So i'd like to design the primer by myself.

I got the genome sequence of DAPK1(Gene bank id:1612) from Gene bank, and found
2 CpG islands in this sequence, but i could not findthe promoter region of the gene, then with
the Genomatix i found 5 promoter regions here and the No.1 CpG island included in two promoter
regions. The location is Chromsome 9:89301936 to 89303914.

So, i used the ABI MSP designer software to analysis the sequence Chromsome 9:89301936 to 89303914,
and design a pair of MSP primer.

My question :
1.Because i've never done this before, so I don't whether there's some problem in this process.
2.The 'hot start' Taq was required in MSP?
3.Could you recommend me some types of genomic DNA Bisulfte modification kit?


Best wishes for you.
Lanbo


--------METHYLATION---------
FORWARD
Length:20 bp.
5' TCGGTTTGGTAGGGTAGTTC 3'
Tm=60.43
CpG=2
C=6

You may modify the primer sequence if necessary, within this region:
5' GTAGAATTCGTAGCGTCGGTTTGGTAGGGTAGTTCGGAGGTGGGTGGGTC 3'
REVERSE
Length:19 bp.
5' AAATCCCCGACGTTAACTC 3'
Tm=60.41
CpG=2
C=TTTGGGTTTCGGAGATTTTAATTTTTTTTTTAGATTGTAAATTTTTTTGTTTTTAAGTTTCGGTTTTAATATTAGTTC
GGTAGAGGAATTTAGTTTAATGAGGTACGTTTTTTTTTTGTTAT4

You may modify the primer sequence if necessary, within this region:
5' TCCGCGAAAAAAACAAAATCCCCGACGTTAACTCGATCCGACTATCCTC 3'
PCR PRODUCT
Length: 167 bp.
5' TCGGTTTGGTAGGGTAGTTCGGAGGTGGGTGGGTCGCGTCGTTAGTTCGTTTGTAGGGTTTTTATTGGTCGTTTGTCGGT
CGTTTTTCGTTTAAAAGGCGGTAAGGAGTCGAGAGGTTGTTTCGGAGTGTGAGGAGGATAGTCGGATCGAGTTAACGTCG
G
GGATTT 3'

--------NO METHYLATION---------

FORWARD
Length: 21 bp.
5' GTTGGTTTGGTAGGGTAGTTT 3'
Tm=58.3

You may modify the primer sequence if necessary, within this region:
5' GTAGAATTTGTAGTGTTGGTTTGGTAGGGTAGTTTGGAGGTGGGTGGGTT 3'
REVERSE
Length: 20 bp.
5' AAAATCCCCAACATTAACTC 3'
Tm=56.88

You may modify the primer sequence if necessary, within this region:
5' TCCACAAAAAAAACAAAATCCCCAACATTAACTCAATCCAACTATCCTC 3'
PCR PRODUCT
Length: 167 bp.
5' TTGGTTTGGTAGGGTAGTTTGGAGGTGGGTGGGTTGTGTTGTTAGTTTGTTTGTAGGGTTTTTATTGGTTGTTTGTTGGT
TGTTTTTTGTTTAAAAGGTGGTAAGGAGTTGAGAGGTTGTTTTGGAGTGTGAGGAGGATAGTTGGATTGAGTTAATGTTG
G
GGATTT 3'

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Hi Lanbo!

I did not completely understand your first question, but I'll try to answer questions 2 and 3. But a stupid question: How have you done the BLAST of your MSP primers? Download the DAPK1 genomic sequence, converted it into bisulfite? Have done the BLAST on both bisulfite-strands? I also sometimes found it difficult to locate MSP-primers of published assays. Use the primers specific for methylated sequences for your BLAST, that's easier. And try to search for part-sequences of your primers. I sometimes observed that there are SNPs in the primer binding-sites!

Of course you need a Taq-polymerase. Hot-Start is the most easiest and safest one I can think of...

I strongly recommend to you the Qiagen 'EpiTect Bisulfite Kit'. It has the highest flexibility regarding sample-type and DNA input amount- and volume. Bisulfite-conversion is excellent. I've never obtained any unconverted DNA. Handling is also very easy. If you have only a very small starting amount of DNA and want to run only one MSP and therefore need a small elution volume to concentrate your sample, use the Zymo 'EZ DNA Methylation Gold Kit'. I've tested nearly all published protocols and commercial available kits and these two are definately the best kits...

Hope that helps. Best,

MoB

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Thank for your answer.
I will BLAST my primer first.

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Try googling methblast.

Nick

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Nice advise! Thank you!

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