How to find promoter sequence for methylation study

Many people have problem identifying or predicting the promoter sequence of a gene, or don't know how to get the actual sequence for analysis such as primer design, transcription factor binding site search, etc. Here I provide ways how I do these things.

1. How to find and retrieve promoter sequences from genome databases

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:

• go to ensembl website: http://www.ensembl.org/index.html
  
• choose an organism such as human http://www.ensembl.o...iens/Info/Index
  
• Search your gene such as BRCA2 http://www.ensembl.o...ns;idx=;q=brca2
  
• 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.o...ns;idx=;q=brca2
  
• On the left, under "Gene Summary", click "Sequence", the sequence of the gene including 5' flanking, exons, introns and flanking region will be displayed.
  
• The exons are high lighted in pink background and red text, the sequence in front of the first exon is the promoter sequence.
  
• 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.
  
• 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
  
• Go to UCSC BLAT search at http://genome.ucsc.e...t?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.
  
• 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.
  
• Beware some genes have alternative promoters. To find those sequences, it requires extensive bioinformatics and experimental analysis.

2. How to predict promoter sequences

... to be updated