6 replies to this topic

[jaa51]

Hi
I am new to this field and would appreciate any help. I am interested in looking at the methylation status of the promoter regions of several genes in a species which has very little sequence information available for it. I have been advised that I need to sequence the promoter regions first. What would be the best way of going about this? There is some sequence information available for only part of the coding sequence for these genes, but nothing more.
Many thanks

[mdfenko]

use the sequence that you know to make a primer that is at least 50 bases away from the region of interest (not more than ~400 bases away).

[phage434]

You can do inverse pcr to amplify regions adjacent to your known region.  You can then sequence those regions.  Repeat as necessary.

[jaa51]

Many thanks for the replies, however I am VERY new to this field, could you clarify a couple of things for me? If I design primers based on the reverse strand and carry out PCR, would I then purify the products and then sequence? Then based on the sequence information for this new strand design new primers and repeat until the area of interest has been covered? Are there specific primer design or Taq guidelines for this type of PCR?
Huge thanks

[phage434]

Inverse PCR relies on cutting genomic DNA with a restriction enzyme, purifying the DNA, then ligating at relatively low concentration to favor the production of circularized molecules.  Outward facing primers are used to amplify across the restriction enzyme site, followed by sequencing.  This yields sequence information for adjacent sequence starting from a region of known sequence.  For the next step, you need to use a different restriction enzyme and new outward facing primers from the region you have just discovered.  This works very well for small bacterial genomes, but I have not tried it on large eukaryotic genomes.

[jaa51]

Thanks for that, much appreciated. One question. How do you choose the correct resitiction enzyme? If the sequence is unknown how can you make sure the RE doesn't cut in the middle of the required sequence?

[phage434]

Well, you can choose based on the statistics of the genome (GC content, mostly), but the point is that you want it to cut, but just not too frequently.  The region you see when sequenced will be the region between the cut site (you don't know where that will be) and the outward directed primer you use.  You'd choose your primer location to avoid cut sites you already know about, or that you intend to use on the next round.