ABI have released MethylPrimer Express! - A new primer design program for DNA methylation....For Free!!! (Jun/02/2006 )

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QUOTE (krümelmonster @ May 28 2007, 11:58 PM)

Hi dongdong,

sorry, I meant the DNA template. To optimize the PCR for the methylated primers it is adivsable to use fully methylated DNA that was bisulfite modified as template.
If your primers (U&M) work efficiently, there are other explanations for your result (no change in detected methylation but different expression). I would mainly guess that it's a methodological problem, as MSP detects only the methylation within the primer binding sites! So, MSP findings do not necessarily reflect the methylation status of the rest of the CpG island. MSP can be used as a screening tool for high-throughput analysis, but the relevance of the CpG's analyzed by your assay has to be confirmed by BSP or pyroseauencing first.
Hope that helps, Krümel

Thanks alot, dear Krümel. I am now trying to do BSP to detect the methylation of each CpG island.

Sincerely,

Dongdong

-dongdong-

Nick,
I have downloaded the program and installed with no problems, but it appears that the program would only recognize true CpG islands and since the gene which I am interested has CpG sites I could not use the program. It appears that I would have to design my primers by conventional programs. Would you have any advice regarding the design of primers (MSP) for real-time PCR

Regards,

Marcos

QUOTE (methylnick @ Jun 2 2006, 08:49 PM)

Hi All,

I figure there will be many people interested in this : check out the link:

Methylprimer Express

And please post comments about this program and if it works for you. I would be interested to know. I am on a Mac and the download isn't working for me.

Cheers

Nick

-marfundo-

Hi MArcos,

methprimer is a program I have seen been used a lot for MSP.

I am sure in methylprimer express you can force a region to amplify and design primers around that.

N

-methylnick-

You can change the settings for CpG island prediction to virtually no CpG present. Than you can select your region fo interest in MPE.

Good luck!

-krümelmonster-

QUOTE (methylnick @ Jun 3 2006, 07:49 AM)

Hi Nick,
Thank you for the information.I released it and designed the MSP primers for PDCD4 with it .Now I don't know the promoter of PDCD4 exactly ,but the the 5' end of the PDCD4 gene has been public in NCBI([AL136368]). I ascertained the 5' end of PDCD4 by aligning its mRNA sequence with sequence AL136368.Then I input sequence within about 500bp upstream and 200bp downstream into the box in the MethylPrimer Express Software,and found one CGi in it.Then design MSP primers as followed.
At first , primers for 147bp amplicon were designed as followed:

MSP -

INITIAL NUCLEOTIDE SEQUENCE

GAGAACCCCAGCACTGGAGGCCGGAATTTGTGCGTTTGAACGCCAGAGGCTTGGCTAGTCATGATTAGCAAAGGAACTTC
AAAGAGTTTTGGAGCCTGCTTTATATCTGTGAAATGGGAATTATTTCCATCTCAAAGGTTTGAGGGGATTCGCTGAGAGAA
ACTTCTTAGCATGGGATCTCCAGAAACTGGAAGCCACAGAGAAAGCAGCGGCCAGAGGGAGGAGGAATCGGACAGGCTGAT
CACTTGGAGTGTTCGCCTAAGCAGTTTCTAGAAATGGAGGCAGGGACCATCAGGAAGCTCAAAACCTTTGCTTCAGCCGAG
TTTGCAGAACGCCCTGTGAGGAGAATGGGTGAGCTGGGTCGAGGAAGCTTCATCCTCGTCCCCATCCCCCAGCACTGCCCT
TTTCCCAACGCTCCCATCCCGCCACGCCTCCCAACATACCCCCACCCCGACTTCCCGCTCAACTCCCGCTCCAGCCAGTCC
CAGGAGCCACATGCGCATGCGCCCTCCCGCGCCCCTCCCCAACTTTCCACGTTTCACTCCTCTCCCTTTTCCTCCTCAGCT
CCGGCTCCGCCGCCACGATTGGCCAGCCGACCACCCGGCCTCGGCCAATAAGCGCCGCCCTCTCGCCCCCGTGTTACTGGG
TAGAAGAAAACAAAAACAAACAGAGCGAGAAGGGCCAGAGACTCTCCGAGGCGGCGGCAGAGACAGAAGAGCG

BISULFITE MODIFICATION OF DNA

GGAAGTTATAGAGAAAGTAGCGGTTAGAGGGAGGAGGAATCGGATAGGTTGATTATTTGGAGTGTTCGTTTAAGTAGTTT
TTAGAAATGGAGGTAGGGATTATTAGGAAGTTTAAAATTTTTGTTTTAGTCGAGTTTGTAGAACGTTTTGTGAGGAGAATG
GGTGAGTTGGGTCGAGGAAGTTTTATTTTCGTTTTTATTTTTTAGTATTGTTTTTTTTTTAACGTTTTTATTTCGTTACGT
TTTTTAATATATTTTTATTTCGATTTTTCGTTTAATTTTCGTTTTAGTTAGTTTTAGGAGTTATATGCGTATGCGTTTTTT
CGCGTTTTTTTTTAATTTTTTACGTTTTATTTTTTTTTTTTTTTTTTTTTAGTTTCGGTTTCGTCGTTACGATTGGTTAGT
CGATTATTCGGTTTCGGTTAATAAGCGTCGTTTTTTCGTTTTCGTGTTATTGGGTAGAAGAAAATAAAAATAAATAGAGCG
AGAAGGGTTAGAGATTTTTCGAGGCGGCGGTAGAGA

METHYLATION
FORWARD

Length:21 bp.
5' GGTCGAGGAAGTTTTATTTTC 3'

Tm=0
CpG=0
C=0

You may modify the primer sequence if necessary, within this region:
5' AGAATGGGTGAGTTGGGTCGAGGAAGTTTTATTTTCGTTTTTATTTTTTAG 3'

REVERSE

Length: 19 bp.
5' CGCATACGCATATAACTCC 3'

Tm=0
CpG=0
C=0

You may modify the primer sequence if necessary, within this region:
5' AAAAACGCGAAAAAACGCATACGCATATAACTCCTAAAACTAACTAAAA 3'

PCR PRODUCT

Length: 147 bp.
5' GGTCGAGGAAGTTTTATTTTCGTTTTTATTTTTTAGTATTGTTTTTTTTTTAACGTTTTTATTTCGTTACGTTTTTTAAT
ATATTTTTATTTCGATTTTTCGTTTAATTTTCGTTTTAGTTAGTTTTAGGAGTTATATGCGTATGCG 3'

NO METHYLATION

FORWARD

Length: 24 bp.
5' TTGGGTTGAGGAAGTTTTATTTTT 3'
Tm=62.03
You may modify the primer sequence if necessary, within this region:
5' AGAATGGGTGAGTTGGGTTGAGGAAGTTTTATTTTTGTTTTTATTTTTTAG 3'

REVERSE

Length: 22 bp.
5' AAACACATACACATATAACTCC 3'

Tm=52.19
You may modify the primer sequence if necessary, within this region:
5' AAAAACACAAAAAAACACATACACATATAACTCCTAAAACTAACTAAAA 3'

PCR PRODUCT

Length: 147 bp.
5' GGTTGAGGAAGTTTTATTTTTGTTTTTATTTTTTAGTATTGTTTTTTTTTTAATGTTTTTATTTTGTTATGTTTTTTAAT
ATATTTTTATTTTGATTTTTTGTTTAATTTTTGTTTTAGTTAGTTTTAGGAGTTATATGTGTATGTG 3'

primers after modified
M: F--5' GGTCGAGGAAGTTTTATTTTC 3'
R--5' CGCATACGCATATAACTCC 3'
U: F--5' GGTTGAGGAAGTTTTATTTTT 3'
R--5' CACATACACATATAACTCC 3'

But my boss told me that it would be better to amplify longer than 147 bp as MSP primers.Maybe 300 to 500bp? I don't know how long is the best for MSP.Could you tell me that ,please?
The primers sites are -235 and +107 ,could this reagion be the promoter of PDCD4?And how about these primers designed?
Thanks very much! Your reply is expected.[or to my email: quanym83@yahoo.com.cn]

-epilab-

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