help - (Aug/18/2007 )

Hi，everyone:
I am a student of Inner Mongolia Agriculture University of China .Now I will do experiment about DNA methylation area ,but I have no any experience at this area and my classmate don’t know this.Now I don’t know who I can ask. So when I see this web today ,I am very glad. At the beginning of experiment, I face some problem that I want to study the DNA methylation of abnormal vertebrae of sheep ,but I don’t know which gene control vertebrae and whether it is methylation region ,I think if I can’t find the gene ,I can’t design the primer.and PCR .I don’t know whether I am right ?if I have some false, please correct me ! I am so sorry that i know nothing abt these..
i would be very greatfull if you could help me out or refer me some papers or books to read about this..
thank you very much!

to dv6000t:

Thank you very much!If I have some problem in my experiment,can i ask you?

Questions regarding specific experimental details are most likely to be answered here. You can also ask very broad questions. However, these are less likely to be addressed. It is your job as a graduate student to study the background information and try to come up with research strategies with the help from your professor/advisor.

hello everyone:
i face some problem in my experiment :i find a gene in genebank ---hoxc8 (X94179) and i want to anlysis hoxc8 of sheep's DNA methylation information ,but this gene(X94179) is about G.gallus ,i can't the sheep 's hoxc8,so i design the primer according this gene using methyl primer express software v1.0,the result is follow.i don't know whether i am correct and there are no one do this topic ,so i really hope someone can help me!if i have some mistake ,please tell me.thank you very much !!

the result of primer designing(yellow is my choice):
INITIAL NUCLEOTIDE SEQUENCE

GAATTCCGTTGCTGTCGGCTCTACCTACCGACAGATTACAAGAGAAGTCAAGAGCACCGGGAACGCACCGTGCGGGCACC
AGAGGCACCATGCAACGCCTTTGAGATCGCCTTTGAAATATATATATTAATAATAATTTTATAATAATAATAAAAAAGCCA
CCTTTTTTATATCTATATATATCTATCTCTCTATTTTAAAAAAAAAAAAAAGACAACAACAACAACAACAAAATAAATAAA
TAAATAAATAATAAAAAAAAGCCGGACTGGTAAACAGAGAGAGAGAGAGAGAGAGAGAGAGAAGAACAGTCCGGGACCGTC
AGCACTCATCCAGAATTAAAGCCGTCTTTTTAATTGTTTTTAATTGTTTTTTTTTTTAATTATTTTTTTCAATTTTTAATT
TTTTTTCAATTTTTATATATATATATGGTTTTTTTTTTTGCATGCGAGGGTCATGAGTTCCTACTTTGTAAATCCTCTCTT
TTCCAAGTACAAAGGCGGCGAATCGCTGGAACCAACTTACTACGACTGCAGATTTCCACAAAGTGTTAGCAGGAGCCATGC
TCTCGTGTATGGTCCGGGCACCACTGCTCCCACCTTCCAGCACCCATCACACCACGTCCAAGAGTTTTTCCACCACGGAAC
CTCCAGCATCTCCAACTCTGGATACCAGCAAAACCCGTGCGCCTTGGCGTGTCACGGAGACGCTTCTAAATTCTATGGATA
TGAAGCTCTGCCGAGGCAATCGCTTTATGGTGCTCAGCAAGAGACGACTGTTGTACAATATCCTGACTGTAAATCGTCTTC
CAACAGTAACTCTAGCGAGGGACAAGGGCATTTAAATCAAAATTCGTCTCCCAGTCTCATGTTCCCATGGATGAGACCTCA
CGCTCCCGGAAGACGCAGTGGCAGACAAACTTACAGCCGGTACCAGACCCTGGAGTTAGAAAAGGAGTTCCTCTTCAACCC
GTATTTGACACGGAAGCGACGGATTGAGGTCTCTCACGCCCTGGGACTGACCGAGAGACAAGTGAAGATCTGGTTCCAGAA
CAGGAGGATGAAGTGGAAAAAAGAAAACAACAAAGATAAGCTGCCGGGGGCCAGAGACGAGGAGAAAACGGAAGAAGAAGG
GAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGCAAGGACTGAGTTCTCAGCCCTTGAAGTCTCGTTTTATG
GCAGATGATAAATCGAGATGTTTACGACGGTCATTTGCTTTTATAGAGTATAGAATGGAGAGGCTCACACAGCAGTAACTA
CCTGTCAAACAGTTGTAGCCTTTTTTATTGTCATAGAAACTTCCCCTACTTTTTGCTTCCCCCCCCCCCCCCACTCTTTTT
GTTTTGTTTTGTTTTGCTTTGCTTTGCTTTTTAATAGCGGCGTTTTTAGCTCCATAAATACATAGTTGGCTTCAGTGCAGC
GACGGTATAAGCGGATGGG

BISULFITE MODIFICATION OF DNA

TTTTTTTTGTATGCGAGGGTTATGAGTTTTTATTTTGTAAATTTTTTTTTTTTTAAGTATAAAGGCGGCGAATCGTTGGA
ATTAATTTATTACGATTGTAGATTTTTATAAAGTGTTAGTAGGAGTTATGTTTTCGTGTATGGTTCGGGTATTATTGTTTT
TATTTTTTAGTATTTATTATATTACGTTTAAGAGTTTTTTTATTACGGAATTTTTAGTATTTTTAATTTTGGATATTAGTA
AAATTCGTGCGTTTTGGCGTGTTACGGAGACGTTTTTAAATTTTATGGATATGAAGTTTTGTCGAGGTAATCGTTTTATGG
TGTTTAGTAAGAGACGATTGTTGTATAATATTTTGATTGTAAATCGTTTTTTAATAGTAATTTTAGCGAGGGATAAGGGTA
TTTAAATTAAAATTCGTTTTTTAGTTTTATGTTTTTATGGATGAGATTTTACGTTTTCGGAAGACGTAGTGGTAGATAAAT
TTATAGTCGGTATTAGATTTTGGAGTTAGAAAAGGAGTTTTTTTTTAATTCGTATTTGATACGGAAGCGACGGATTGAGGT
TTTTTACGTTTTGGGATTGATCGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAA
TAAAGATAAGTTGTCGGGGGTTAGAGACGAGGAGAAAACGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGA
AAAAGAAGAAAGTAAGGATTGAGTTTTTAGTTTTTGAAGT

FORWARD

Length: 23bp.
5' TTGGAGTTAGAAAAGGAGTTTTT 3'
Tm=58.6; CpG=0; C=3
You may modify the primer sequence if necessary, within this region:
5' AGTYGGTATTAGATTTTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTT 3'


REVERSE

Length: 25 bp.
5' TCAAAAACTAAAAACTCAATCCTTA 3'
Tm=58.46; CpG=0; C=7
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTTCC 3'


PCR PRODUCT

Length: 261 bp.
5' TTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGGATT
GATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTYGGG
GGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAAGGA
TTGAGTTTTTAGTTTTTGA 3'

%CGs=35.25




ADDITIONAL PRIMERS

NUMBER 1(937,958) -- (1179,1200)

FORWARD

Length:22 bp.
5' ATTTTGGAGTTAGAAAAGGAGT 3'
Tm=56.43; CpG=0; C=3

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

REVERSE

Length:22 bp.
5' TCAAAAACTAAAAACTCAATCC 3' Tm=56.38; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTT 3'

PCR PRODUCT

Length: 264 bp.
5' ATTTTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGG
ATTGATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTY
GGGGGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAA
GGATTGAGTTTTTAGTTTTTGA 3'

%CGs=34.85


NUMBER 2(940,961) -- (1178,1200)

FORWARD

Length:22 bp.
5' TTGGAGTTAGAAAAGGAGTTTT 3'
Tm=57.41; CpG=0; C=3

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

REVERSE

Length:23 bp.
5' TCAAAAACTAAAAACTCAATCCT 3' Tm=57.31; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTT 3'

PCR PRODUCT

Length: 261 bp.
5' TTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGGATT
GATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTYGGG
GGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAAGGA
TTGAGTTTTTAGTTTTTGA 3'

%CGs=35.25

NUMBER 3(940,962) -- (1177,1200)

FORWARD

Length:23 bp.
5' TTGGAGTTAGAAAAGGAGTTTTT 3'
Tm=58.6; CpG=0; C=3

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

REVERSE

Length:24 bp.
5' TCAAAAACTAAAAACTCAATCCTT 3' Tm=58.48; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTTC 3'

PCR PRODUCT

Length: 261 bp.
5' TTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGGATT
GATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTYGGG
GGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAAGGA
TTGAGTTTTTAGTTTTTGA 3'

%CGs=35.25


NUMBER 4(934,956) -- (1178,1200)

FORWARD

Length:23 bp.
5' TAGATTTTGGAGTTAGAAAAGGA 3'
Tm=57.39; CpG=0; C=4

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

REVERSE

Length:23 bp.
5' TCAAAAACTAAAAACTCAATCCT 3' Tm=57.31; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTT 3'

PCR PRODUCT

Length: 267 bp.
5' TAGATTTTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTT
GGGATTGATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTT
GTYGGGGGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAG
TAAGGATTGAGTTTTTAGTTTTTGA 3'

%CGs=34.83


NUMBER 5(940,961) -- (1168,1191)

FORWARD

Length:22 bp.
5' TTGGAGTTAGAAAAGGAGTTTT 3'
Tm=57.41; CpG=0; C=3

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

REVERSE

Length:24 bp.
5' AAAAACTCAATCCTTACTTTCTTC 3' Tm=57.34; CpG=0; C=4
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTTCCTCCTCTTC 3'

PCR PRODUCT

Length: 252 bp.
5' TTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGGATT
GATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTYGGG
GGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAAGGA
TTGAGTTTTT 3'

%CGs=35.71


NUMBER 6(940,961) -- (1179,1200)

FORWARD

Length:22 bp.
5' TTGGAGTTAGAAAAGGAGTTTT 3'
Tm=57.41; CpG=0; C=3

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

REVERSE

Length:22 bp.
5' TCAAAAACTAAAAACTCAATCC 3' Tm=56.38; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTT 3'

PCR PRODUCT

Length: 261 bp.
5' TTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGGATT
GATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTYGGG
GGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAAGGA
TTGAGTTTTTAGTTTTTGA 3'

%CGs=35.25


NUMBER 7(937,959) -- (1178,1200)

FORWARD

Length:23 bp.
5' ATTTTGGAGTTAGAAAAGGAGTT 3'
Tm=57.67; CpG=0; C=3

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

REVERSE

Length:23 bp.
5' TCAAAAACTAAAAACTCAATCCT 3' Tm=57.31; CpG=0; C=6
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTT 3'

PCR PRODUCT

Length: 264 bp.
5' ATTTTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTTGGG
ATTGATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTTGTY
GGGGGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAGTAA
GGATTGAGTTTTTAGTTTTTGA 3'

%CGs=34.85


NUMBER 9(934,956) -- (1168,1191)

FORWARD

Length:23 bp.
5' TAGATTTTGGAGTTAGAAAAGGA 3'
Tm=57.39; CpG=0; C=4

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

REVERSE

Length:24 bp.
5' AAAAACTCAATCCTTACTTTCTTC 3' Tm=57.34; CpG=0; C=4
You may modify the primer sequence if necessary, within this region:
5' ACTTCAAAAACTAAAAACTCAATCCTTACTTTCTTCTTTTTCCTCCTCTTC 3'

PCR PRODUCT

Length: 258 bp.
5' TAGATTTTGGAGTTAGAAAAGGAGTTTTTTTTTAATTYGTATTTGATAYGGAAGYGAYGGATTGAGGTTTTTTAYGTTTT
GGGATTGATYGAGAGATAAGTGAAGATTTGGTTTTAGAATAGGAGGATGAAGTGGAAAAAAGAAAATAATAAAGATAAGTT
GTYGGGGGTTAGAGAYGAGGAGAAAAYGGAAGAAGAAGGGAATGAGGAAGAGGAAAAAGAAGAGGAGGAAAAAGAAGAAAG
TAAGGATTGAGTTTTT 3'

%CGs=35.27


NUMBER 10(451,473) -- (863,885)

FORWARD

Length:23 bp.
5' AGGGTTATGAGTTTTTATTTTGT 3'
Tm=56.17; CpG=0; C=4

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

REVERSE

Length:23 bp.
5' TCTCATCCATAAAAACATAAAAC 3' Tm=56.15; CpG=0; C=5
You may modify the primer sequence if necessary, within this region:
5' TCCRAAAACRTAAAATCTCATCCATAAAAACATAAAACTAAAAAACRAATTTT 3'

PCR PRODUCT

Length: 435 bp.
5' AGGGTTATGAGTTTTTATTTTGTAAATTTTTTTTTTTTTAAGTATAAAGGYGGYGAATYGTTGGAATTAATTTATTAYGA
TTGTAGATTTTTATAAAGTGTTAGTAGGAGTTATGTTTTYGTGTATGGTTYGGGTATTATTGTTTTTATTTTTTAGTATTT
ATTATATTAYGTTTAAGAGTTTTTTTATTAYGGAATTTTTAGTATTTTTAATTTTGGATATTAGTAAAATTYGTGYGTTTT
GGYGTGTTAYGGAGAYGTTTTTAAATTTTATGGATATGAAGTTTTGTYGAGGTAATYGTTTTATGGTGTTTAGTAAGAGAY
GATTGTTGTATAATATTTTGATTGTAAATYGTTTTTTAATAGTAATTTTAGYGAGGGATAAGGGTATTTAAATTAAAATTY
GTTTTTTAGTTTTATGTTTTTATGGATGAGA 3'

%CGs=25.29

yonggi,

ideally you would want to know the hoxc8 gene sequence in sheep, I am sure there are genome sequences for this. try genome browser. i suspect this could be a highly conserved gene and MAYNOT be different to the mouse but i am not sure.

you need to know the sequence to design proper primers for methylation analysis.

Nick

Hello! everyone:
In my experiment, I face a problem :After I modify my genomic DNA,I detect it by agarose gel electrophoresis ,then stain by EB,but there are no band on the gel.Does it mean that modification is not succeed or it cann't be detected because DNA is single chain? I follow the general method to modify my genomic DNA and now I don't know it is because the promblem or other reason.So I hope someone can help me .Thanks in advance!

yongyi, if you are using the conventional method, you won't be able to see a band after conversion, because you would have much less converted DNA than what you started with. you need to perform the PCR and look for the amplicon.

Nick