Amplifying a gene using a degenerate primer - (Jun/03/2009 )

Dear all,

Cutting through all the steps in designing degenerate primers, usually conserved regions of our target gene occur well into the middle i.e. for a 600 amino acid gene, conserved regions may appear, say, beginning at amino acid 100.

What happens during PCR is that the primer will bind (hopefully) and amplify only the inside region of the gene. Even if we get a product, it wouldn't be of use. One would tend to say, "Hey, sequence it and design a specific primer". Alas, my sequence will begin in the "middle" as well since the region upstream from where my primer bind to isn't amplified.


To present it visually, look at the Imaginary gene below and the region to which the primers bind to (in bold).

.....ATGGCGCATGCTGATGCATATGGCAGGCGCC.....

The region outside the primers will be lost during amplification so how am I going to obtain my gene and it's complete sequence? By using a labelled probe?

Thanks people!

You can try genomic sequencing (sequencing directly from the chromosome) -- some sequencing centers can do this. Two sequencing reactions, one with each of your two primers, might cover your entire gene.

You can use your PCR product as a probe against a Southern blot of your chromosome digested with several restriction enzymes individually to try and find your gene on a reasonably sized fragment you can clone.

You can make a library of cosmid clones, and probe it with your PCR product via colony blots to find a clone containing your gene.

What organism is your gene from? You might be able to find a genomic sequence of it, and save yourself a lot of trouble.

This is a perfect application for inverse pcr. The idea is to cut your DNA with a restriction enzyme that frequently cuts (Sau3AI, for example), dilute the DNA, and then religate. At high dilutions, circular products are preferentially formed. You can now PCR from those circular products using primers directed outward from your known sequence. Sequencing these pcr products provides additional flanking sequence surrounding your known sequence, and you can identify the restriction site to tell you where the splice between left and right sequence occurs. You can repeat with a different enzyme to get additional coverage.

you can also make a RACE with specific primers designed on your fragment

Gosh. Very useful direction. Thanks guys!