|
This is the URL to my gene of interest. I am trying to estimate the promoter region of this gene so that I can do Meth specific PCR on the promoter..the promoter region of this gene has not been positively identified..but the exon information is known. I am new to this field.. Could anyone please help me out, preferably using step by step directions if possible. Thank you!!! -eagertolearn- -------------------------------------------------------------------------------- Here is what I did to find the 5' flanking sequence of your gene: 1. I searched NCBI nucleotide database using "gene name + promoter" or "gene name + flanking" and got no hit. 2. Then, I went to ensembl.org and found the gene, clicked "View genomic sequence for this gene with exons highlighted", and asked it to export 1kb 5' sequence. The new Ensembl website has moved this link to the left side and the link is called Genomic Sequence 3. I copied the 5' sequence and fed to methprimer to predict CpG islands. Luckily, there is a CpG island close to the first exon (see fig below), which means the 5' sequence is likely the promoter of the gene. 4. I pasted the sequence back to NCBI blastn program to see if it matches experimentally identified promoter sequences that I might have missed by gene name text search. In this case, I didn't find any, which means the promoter of this gene has not been characterized yet. You can safely assume the sequence I got is the 5' regulatroy sequence and go ahead with methylation mapping of the identified CpG island. Hope that helps. 5' Flanking sequence (red is exon): GAAGGCCAAGTACCCCAACTCTCCCCGACGCTGCTGAGCCCTTCCCTCCCCTAATCTGAG CpG island prediction (blue) and BSP primer design -pcrman- -------------------------------------------------------------------------------- Thank you very much!! I have one more query..Is there a way to do this using the Entrez gateway? The problem is my boss trusts ncbi more than anything else..thank you very much again! -eagertolearn- -------------------------------------------------------------------------------- You can just blast NCBI database using the sequence I got from ensembl. I am sure this sequence and the mRNA sequence will align to the same genomic sequence. Actually all genome databases (ensembl, UCSC and NCBI) use the same data and assembly but present the data differently. ensembl provides the best genome browser. -pcrman- -------------------------------------------------------------------------------- Hi pcrman, How do you determine which sequence is the exon and whether it is exon 1 or 2 or so on? -mrpcr- -------------------------------------------------------------------------------- All genome databases provide gene structure information. The best place to look for that is Ensembl. -pcrman- -------------------------------------------------------------------------------- Hi, PCR man: I am new to this field. I am trying to design MSP primers for rat c-myc so I read all of your post. How did you know the the promoter region is in the 1kb sequence before the first exon? Thanks! -hn37041- -------------------------------------------------------------------------------- Most genes if not all have their promoter immediately upstream the first exon. -pcrman- -------------------------------------------------------------------------------- Hi, pcrman i am a new one here. i wanna study the methlyation status in the promoter of human GFAP in glioma cells so i read almost posts here.Followed your direction step by step,i tried to find CpG islands and to design BSP or MSP primer of this gene,but the results puzzled me.Could you help me out? At first i searched NCBI and got a sequence--"Homo sapiens glial fibrillary acidic protein (GFAP), mRNA,3035bp, NM_002055".then i fed this 3035bp sequence to metheprimer,there is a CpG islands from 649 to 1142,and BSP primer were as follows: Primer picking results for bisulfite sequencing (or restriction) PCR Primer Start Size Tm GC% 'C's Sequence 1 Left primer 787 25 51.35 52.00 7 GTTTTAAGTTTGTAGATTTGATAGA Right primer 1030 23 57.22 60.87 4 TTAAAACTCTACCCCTCTTCCTC Product size: 244, Tm: 73.2, CpGs in product: 23 2 Left primer 787 25 51.35 52.00 7 GTTTTAAGTTTGTAGATTTGATAGA Right primer 1031 24 58.14 62.50 4 CTTAAAACTCTACCCCTCTTCCTC Product size: 245, Tm: 73.2, CpGs in product: 23 3 Left primer 787 25 51.35 52.00 7 GTTTTAAGTTTGTAGATTTGATAGA Right primer 1032 24 59.60 62.50 4 CCTTAAAACTCTACCCCTCTTCCT Product size: 246, Tm: 73.3, CpGs in product: 23 4 Left primer 787 25 51.35 52.00 7 GTTTTAAGTTTGTAGATTTGATAGA Right primer 1029 23 59.18 65.22 4 TAAAACTCTACCCCTCTTCCTCC Product size: 243, Tm: 73.3, CpGs in product: 23 5 Left primer 787 25 51.35 52.00 7 GTTTTAAGTTTGTAGATTTGATAGA Right primer 1033 23 57.22 60.87 4 TCCTTAAAACTCTACCCCTCTTC Product size: 247, Tm: 73.2, CpGs in product: 23 Then I wanted to identify this result so i went to ensembl.org and found the gene, clicked "View genomic sequence for this gene with exons highlighted", and asked it to export 1kb 5' sequence.the result as follows: CAGCCAGCTCATGTGTAACGGCTTTGTGGAGCTGTCAAGGCCTGGTCTCTGGGAGAGAGGCACAGGGAGGCCAGACAAGGAAGGGGTGACCTGGAGGGACAGATCCAGGGGCTAAAGTCCTGATAAGGCAAGAGAGTGCCGGCCCCCTCTTGCCCTATCAGGACCTCCACTGCCACATAGAGGCCATGATTGACCCTTAGACAAAGGGCTGGTGTCCAATCCCAGCCCCCAGCCCCAGAACTCCAGGGAATGAATGGGCAGAGAGCAGGAATGTGGGACATCTGTGTTCAAGGGAAGGACTCCAGGAGTCTGCTGGGAATGAGGCCTAGTAGGAAATGAGGTGGCCCTTGAGGGTACAGAACAGGT TCATTCTTCGCCAAATTCCCAGCACCTTGCAGGCACTTACAGCTGAGTGAGATAATGCCTGGGTTATGAAATCAAAAAGTTGGAAAGCAGGTCAGAGGTCATCTGGTACAGCCCTTCCTTCCCTTTTTTTTTTTTTTTTTTGTGAGACAAGGTCTCTCTCTGTTGCCCAGGCTGGAGTGGCGCAAACACAGCTCACTGCAGCCTCAACCTACTGGGCTCAAGCAATCCTCCAGCCTCAGCCTCCCAAAGTGCTGGGATTACAAGCATGAGCCACCCCACTCAGCCCTTTCCTTCCTTTTTAATTGATGCATAATAATTGTAAGTATTCATCATGGTCCAACCAACCCTTTCTTGACCCACCTTCCTAGAGAGAGGGTCCTCTTGCTTCAGCGGTCAGGGCCCCAGACCCATGGTCTGGCTCCAGGTACCACCTGCCTCATGCAGGAGTTGGCGTGCCCAGGAAGCTCTGCCTCTGGGCACAGTGACCTCAGTGGGGTGAGGGGAGCTCTCCCCATAGCTGGGCTGCGGCCCAACCCCACCCCCTCAGGCTATGCCAGGGGGTGTTGCCAGGGGCACCCGGGCATCGCCAGTCTAGCCCACTCCTTCATAAAGCCCTCGCATCCCAGGAGCGAGCAGAGCCAGAGCAGGATGGAG i fed this sequence to methprimer.However,no CpG island was found,and BSP primers were changed: Primer Start Size Tm GC% 'C's Sequence 1 Left primer 861 25 59.31 64.00 7 GGTGAGGGGAGTTTTTTTTATAGTT Right primer 1020 24 52.67 62.50 4 CTCCATCCTACTCTAACTCTACTC Product size: 160, Tm: 71.0, CpGs in product: 5 2 Left primer 862 25 58.66 64.00 7 GTGAGGGGAGTTTTTTTTATAGTTG Right primer 1020 24 52.67 62.50 4 CTCCATCCTACTCTAACTCTACTC Product size: 159, Tm: 71.1, CpGs in product: 5 3 Left primer 819 24 58.29 66.67 8 GTGTTTAGGAAGTTTTGTTTTTGG Right primer 1020 24 52.67 62.50 4 CTCCATCCTACTCTAACTCTACTC Product size: 202, Tm: 70.5, CpGs in product: 5 4 Left primer 863 24 57.93 62.50 7 TGAGGGGAGTTTTTTTTATAGTTG Right primer 1020 24 52.67 62.50 4 CTCCATCCTACTCTAACTCTACTC Product size: 158, Tm: 71.2, CpGs in product: 5 5 Left primer 820 23 57.51 65.22 8 TGTTTAGGAAGTTTTGTTTTTGG Right primer 1020 24 52.67 62.50 4 CTCCATCCTACTCTAACTCTACTC Product size: 201, Tm: 70.6, CpGs in product: 5 Now i do not know which result is correct? And which primer pair above can i select is best to my study? Thanks in advance for your help. wenyaya -wenyaya- -------------------------------------------------------------------------------- Hi wenyaya, Sorry for the late reply. You are right there is no CpG island in the 5' flanking region (up to 1kb) of GFAP gene and even in the first exon and intron, although the first exon is relatively GC-rich. In this case if I were you I would not expect its methylation has an impact on transcription and thus would not analyze its methylation for disease association. -pcrman- -------------------------------------------------------------------------------- not too sure why you chose 340bp upstream of the gene. I routinely use , it's easier to navigate the database than NCBI and ensembl is some ways. I have attached the grapical readout of this gene, there is indeed a CpG islands at the 5' end. As for the differences between ensembl and NCBI, I am not too sure about that one. Good luck nick -methylnick- -------------------------------------------------------------------------------- Hi,I am very glad to see posts about methylation protocol here. I have already tried in this aspect for a long time.I bought a kit about p16 ,modification and methylation.And I tried with Human samples.I have gotten very good results,and the bands were very clear.Then I tried with mouse samples,extracted DNA from mouse spleen,modificated and amplified with the primer designed like pcrman mentioned.But no bands appeared.And I tried with more primers like p15,pten,etc,still no band. Could you please tell me what is my problem? Comparing with the kit for human(no kit for mouse),there is only difference with primers.I wonder if I should modificate the primer designed with methprimer? I am a new postdoc here and I just can not do this well.I am very hopeful for your help.Thanks! -LISA- -------------------------------------------------------------------------------- |
→如果您认为本词条还有待完善，请 编辑词条
上一篇Frozen competent E. coli cells 【Carnegie Institution of Washington】 下一篇MITOCHONDRIAL?DNA?ISOLATION
词条内容仅供参考，如果您需要解决具体问题
（尤其在法律、医学等领域），建议您咨询相关领域专业人士。
0
收藏到: 
