Isolating a specific gene (specifically TRAV* series of genes) for sequencing

Question

I'm trying to figure out (for pedagogical purposes) the right way to isolate and PCR the regions coding the T-Cell receptor.

My understanding is that I would need to use restriction enzymes that target regions near the start and end of the gene. I was looking at a bunch of different restriction enzymes on this site: https://www.addgene.org/mol-bio-reference/restriction-enzymes/

What I'm not sure about is: how do you pick the combination that will specifically cut out that gene and not any of the other ones? It seems like the restriction enzymes only consist of a few base pairs, and I would guess they can slice at many many sites.

Furthermore once it is cut, how do I isolate just the DNA for the gene I want to sequence as opposed to the rest of the junk DNA?

Answer 1

Restriction enzymes are usually only used when you do molecular cloning using plasmids or other forms of shorter DNA moleclues, since - like you said - using them on genomic DNA would cut it into much more than thousands of pieces.

Instead what you can do is perform your PCR directly on the genomic DNA (or potentially cDNA, if you want to get rid of introns). Doing PCR on genomic DNA is often a bit tricky (i.e. you'll likely have to try different conditions to get a product), but generally possible. If you want to analyse the whole coding region it might be better to extract mRNA instead of DNA and reverse transcribe it, so that can amplify the coding region as one block, that doesn't contain Introns - PCR on cDNA is also easier than on genomic DNA.

Another thing you need to look out for is that T-Cell-Receptors (similar to antibodies) are different in every T-Cell. This means that you can't use Sanger sequencing, which will only give you a proper result if you input has the exact same sequence. To get the sequences of a mix of T-cells you would need to use next-gen sequencing.