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