Amplifying a gene using a degenerate primer
Posted 02 June 2009 - 09:41 PM
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).
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?
Posted 03 June 2009 - 02:12 AM
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.
Posted 03 June 2009 - 03:58 AM
Posted 04 June 2009 - 12:39 PM