How to find the promoter - (Oct/02/2009 )

I want to do methylation specific PCR. At first, I need to search the gene's promoter to design the probe. I find the mRNA sequence in NCBI, then the DNA sequence. Interestedly, the mRNA sequence is just located in the origination of the DNA sequence.
Usually, we find the promoter in the transcription start site upstream 2000bp. But i can't find any sequence before DNA except mRNA.
Would u please tell me the search protocol step by step? I am newer in this field. Any suggestions or guidance would be much appreciated. Thanks a lot!
Here is my gene's mRNA.
i|17999523|ref|NM_032489.2| Homo sapiens acrosin binding protein (ACRBP), mRNA
TCCTTCCCTCACTCCTGAAGGTGCTGCTCCTGCCTCTGGCACCTGCCGCAGCCCAGGATTCGACTCAGGC
CTCCACTCCAGGCAGCCCTCTCTCTCCTACCGAATACGAACGCTTCTTCGCACTGCTGACTCCAACCTGG
AAGGCAGAGACTACCTGCCGTCTCCGTGCAACCCACGGCTGCCGGAATCCCACACTCGTCCAGCTGGACC
AATATGAAAACCACGGCTTAGTGCCCGATGGTGCTGTCTGCTCCAACCTCCCTTATGCCTCCTGGTTTGA
GTCTTTCTGCCAGTTCACTCACTACCGTTGCTCCAACCACGTCTACTATGCCAAGAGAGTCCTGTGTTCC
CAGCCAGTCTCTATTCTCTCACCTAACACTCTCAAGGAGATAGAAGCTTCAGCTGAAGTCTCACCCACCA
CGATGACCTCCCCCATCTCACCCCACTTCACAGTGACAGAACGCCAGACCTTCCAGCCCTGGCCTGAGAG
GCTCAGCAACAACGTGGAAGAGCTCCTACAATCCTCCTTGTCCCTGGGAGGCCAGGAGCAAGCGCCAGAG
CACAAGCAGGAGCAAGGAGTGGAGCACAGGCAGGAGCCGACACAAGAACACAAGCAGGAAGAGGGGCAGA
AACAGGAAGAGCAAGAAGAGGAACAGGAAGAGGAGGGAAAGCAGGAAGAAGGACAGGGGACTAAGGAGGG
ACGGGAGGCTGTGTCTCAGCTGCAGACAGACTCAGAGCCCAAGTTTCACTCTGAATCTCTATCTTCTAAC
CCTTCCTCTTTTGCTCCCCGGGTACGAGAAGTAGAGTCTACTCCTATGATAATGGAGAACATCCAGGAGC
TCATTCGATCAGCCCAGGAAATAGATGAAATGAATGAAATATATGATGAGAACTCCTACTGGAGAAACCA
AAACCCTGGCAGCCTCCTGCAGCTGCCCCACACAGAGGCCTTGCTGGTGCTGTGCTATTCGATCGTGGAG
AATACCTGCATCATAACCCCCACAGCCAAGGCCTGGAAGTACATGGAGGAGGAGATCCTTGGTTTCGGGA
AGTCGGTCTGTGACAGCCTTGGGCGGCGACACATGTCTACCTGTGCCCTCTGTGACTTCTGCTCCTTGAA
GCTGGAGCAGTGCCACTCAGAGGCCAGCCTGCAGCGGCAACAATGCGACACCTCCCACAAGACTCCCTTT
GTCAGCCCCTTGCTTGCCTCCCAGAGCCTGTCCATCGGCAACCAGGTAGGGTCCCCAGAATCAGGCCGCT
TTTACGGGCTGGATTTGTACGGTGGGCTCCACATGGACTTCTGGTGTGCCCGGCTTGCCACGAAAGGCTG
TGAAGATGTCCGAGTCTCTGGGTGGCTCCAGACTGAGTTCCTTAGCTTCCAGGATGGGGATTTCCCTACC
AAGATTTGTGACACAGACTATATCCAGTACCCAAACTACTGTTCCTTCAAAAGCCAGCAGTGTCTGATGA
GAAACCGCAATCGGAAGGTGTCCCGCATGAGATGTCTGCAGAATGAGACTTACAGTGCGCTGAGCCCTGG
CAAAAGTGAGGACGTTGTGCTTCGATGGAGCCAGGAGTTCAGCACCTTGACTCTAGGCCAGTTCGGATGA
GCTGGCGTCTATTCTGCCCACACCCCAGCCCAACCTGCCCACGTTCTCTATTGTTTTGAGACCCCATTGC
TTTCAGGCTGCCCCTTCTGGGTCTGTTACTCGGCCCCTACTCACATTTCCTTGGGTTGGAGCAACAGTCC
CAGAGAGGGCCACGGTGGGAGCTGCGCCCTCCTTAAAAGATGACTTTACATAAAATGTTGATCTTC

Hiiii,

Promoter sequences are usually the sequence immediately upstream the transcription start site (TSS) or first exon. If we know the TSS of a gene, we will know with confidence where the promoter is even without experimental characterization. For many organisms, such as as human, mouse, the genome is well annotated and TSS well defined. Thus promoter sequence retrieval is an easy task. There are three major genome browsers: NCBI, Ensembl and UCSC. For our purpose, Ensembl provides the most convenient interface. Here is an example:


1 go to ensembl website: http://www.ensembl.org/index.html

2 choose an organism such as human http://www.ensembl.org/Homo_sapiens/Info/Index

3 Search your gene such as BRCA2 http://www.ensembl.org/Homo_sapiens/Search...ns;idx=;q=brca2

4 Click the right hit on the search result page and it will bring you to the gene summary page. For example the link to BRCA2 gene is http://www.ensembl.org/Homo_sapiens/Search...ns;idx=;q=brca2

5 On the left, under "Gene Summary", click "Sequence", the sequence of the gene including 5' flanking, exons, introns and flanking region will be displayed.

6 The exons are high lighted in pink background and red text, the sequence in front of the first exon is the promoter sequence.

7 By default, 600 bp 5'-flanking sequence (promoter) is displayed. If you want to get more, click "Configure this page" in the lower left column, a popup window opens allowing to input the size of 5' Flanking sequence (upstream). You can put for example "1000" and then save the configuration.

8 Sometimes there are discrepancies between Ensembl and UCSC annotation regarding TSS. To make sure the first exon given by ensembl is right, copy the promoter sequence

9 Go to UCSC BLAT search at http://genome.ucsc.edu/cgi-bin/hgBlat?command=start and choose the right genome (eg, human), paste the sequence there. On the result page, click browse of the first hit, this will bring you to the genome browser Page. the query sequence is now aligned with UCSC genome sequence. Zoom out a bit, you will be able to determine whether the promoter sequence matches UCSC annotation. If it matches, the sequence is very likely the right one. Here is the BRCA2 promoter sequence aligned to BRAC2 gene .

10 In UCSC genome broswer, you can turn on CpG island feature, if there is CpG island in the promoter sequence, the sequence is highly likely a true promoter. In the above example (BRCA2), a CpG island is displayed in the proximal promoter.

11 Beware some genes have alternative promoters. To find those sequences, it requires extensive bioinformatics and experimental analysis.

MENTIONED BELOW ARE THE SEQUENCE WHICH I DEDUCED USING YR mRNA ref sequence

Promoter sequence:
GACGAGGTTTCGCCCTGTTGCCCAGGCTGGAATTTCTCATTTTAAACACAAAACAGATAC
AAAAGCAAAAGTTCTCCTCCCATACCTCAGTGGACTGTCTTACTCGATTCTCCCTTGGAA
ACCACTTTCGTAGACCACTGAGCCACCCTCTTGTTTTCCCCTTGGACCCATTTTGCTCAT
GGAGGCGCCCTGTCTAGTTCCTACCCAGATAGGCCTTGACTTGGTTTTGCACATTCTATC
TCTGCCTGCGCTTAGCTCTAGACCTCACGACCTGTTTGTTGGGCGAGTTGCTTCCCTAAG
CCCCGATTTCTCATCTGTAAAACGAATGATAGTGTATCTACCTTATGGGATTCCTATGGC
AATTACGTAAGGTAGTGCATGTAAAGATGCTAAGTGGCTGGCTACGGTGGCTCACACCTG
TAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCATTTGAAGTCAGGAGTTCAAGAC
CAGCCTGGCCAACATGGTGAAACCCAGTCTCTACTAAAAATACAAAAATTAGGCCGGGCA
TGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCGGATCACAAGGT
CAAGAGATCGAGACCATCTGGCCAGCATGGTGAAATACCATCTCTACTAAAAATACAAAA
ATTAGCTGGGCGTGGTGGCGTGCACCTATAGTCCCAGCTACTCGGGAGGCTGAGGCAGGA
GAATCACTTGAACCTGGGACGCCGAGGTTGTAGTGAGCCGAGATCGCACCACTGCACTCC
AGCCTGGGCGACAGAGTAGGACTCCGTCTCAAAAAAAAAAGAAAAAGACGCTAAGTGAAG
GTAGCTGTTTGCCATTCCTACCATCTGGATGGGCTCTGGCCACTTTAGGGCCCTGAGAGC
CCGGCTGTCGAGGCCCCGCCCCGGCCCGCTCTTTGTGACGCGTGGGCGGTGCCCGCGTGC
GCCCCGCCCCGCGCCTGCGGCTCTCTCTGCGGCTTGGCCC

Exon 1
GTTAGAGGCGGCTTGTGTCCACGGGACGCGGGCGGATCTTCTCCGGCCATGAGGAAGCCAGCCGCTGGCTTCCTTCCCTC


just copy paste the link u will get the entire sequence

http://www.ensembl.org/Homo_sapiens/Gene/S...ENSG00000111644

Hope this is info is helpful to you

Cheers :-}

Firoz

glbinbin on Oct 2 2009, 05:55 PM said:

Hi Firoz,

Its a good description. Could you please recommend me some useful resource to find promoter for any gene of Arabidopsis thaliana?

thanks

Thanks! I got it!

Hi
I have no idea abut plant genome.

Thanks,
FA

Signal on Oct 4 2009, 01:25 AM said:

Hi firoz,

thanks for the detailed description - been digging ma brains out on trying to find a suitable promoter prediction program - but i dont quite understand the alignment of the possible promoter sequence obtained from ensembl with the UCSC BLAT search. what do u mean by "Zoom out a bit, you will be able to determine whether the promoter sequence matches UCSC annotation".

I am specifically looking for the promoter of the human SLC22A1 gene mapped onto chromosome 6. COuld you please help me out with this?

Hi
Can u send me the reference acession no of yr gene SLC22A1, I mean is it NM_003057 or something else??

If it is NM_003075 then mentioned below is the promoter sequence

>hg18_refGene_NM_003057 range=chr6:160461853-160499604 5'pad=0 3'pad=0 strand=+ repeatMasking=none

tccttactcacctcacgtgtagatctgttttcagtagtcatcactacatg
ttataatggtagctcttagtaattgttaattttggtgaaatacccagtaa
gttattttagttacgtacttaggtgcagatgaggtctaactatttccagc
atagctagggcaggccctaccaaactgcaaagcaggaaagttgaacaatt
ttcaaaagccaaagcagtttatgaccttaaagcatttagtcaacctagtt
tctgacttgcataatttagaccatgtctatattttgaagacatttctatt
ttcattttactgataatttaaaagacagcctatttagcaaagtcatactt
aagtgaatttgaaaattgcttagacttatttacttaatttatgaacactc
ttttacttataagccaatttggtagacacaacatataacagtaagtgtac
atacaagtaaacacatctagacatgtatacacacacacaaatgaagaggt
ggagcttaaaacacccaaattgggtgcagtgtatactgcttgggagatgg
atgcaccaaaatctcacaaatcaccactaaagaacctgctcatgtaacca
aataatacctgttccctcaaaacctatgggaaaaaaaaactaaaaaaaat
acttaaatggtatcacagaactaattagccgaatacagtatctagtacct
ggctgtcacccaatacttgcctcataccatcacatctagaaaacaagtag
atattctttttggaagagccctgagggagctactaggaggtttgcacggc
ctgctctcctgccctcttcttgctctgtggctgaacttcaattctcttcg
ggcttagaccccactgactcgctcccgggcaaagcaaacgatttgatcag
atggccacgtgcattcttccttttcctgaaaccagcaccatagggtaaaa
gattatttctacttggttgccttccagatgtttcacacttggacagcaaa
ctgatttcaaaccactccttttcaaagatctctgagggagacattgcacc
tggccactgcagcccagagcaggtctggccacggccatgagcatgctgag
ccatc
ATGCCCACCGTGGATGACATTCTGGAGCAGGTTGGGGAGTCTGGC: CDS

I have got this sequence from UCSC genome browser, the details of the same can be viewed at

http://genome.ucsc.edu/cgi-bin/hgc?hgsid=1...p;submit=submit

sandi on Oct 6 2009, 01:43 AM said:

Thank u so much Firoz -