Checking for mutations

How can PCR and DNA sequencing be used to identify whether a specific mutation is present or not?

Transcript

Wellington College Don, can you tell us about some of the techniques that use in order to investigate that?

Dr Love Auckland University So let’s say there is a mutation, I want to impose a defined mutation event on a gene. How do I know I’ve done that? I need to amplify essentially a small region of that zebrafish genome and interrogate that little bit because that’s the bit where I think my mutation should be. So I need to amplify it from (using the current vernacular) the bugger all amounts that I’ve got, to reveal enough that I can now sequence it.

So in the human genome, your haploid genome equivalent is 3,000 mega base pairs, or 3 x 10 9 base pairs. But I’m only interested in 100 base pairs of that, let’s say. I am only interested in that because in that little bit I want to know whether there is a mutation event, a critical mutation event.

Let’s say it’s an autosomal copy. You’ve got two copies of this gene, one of which is a mutant copy. I amplify a little region of this gene which is pivotal, and I’m going to sequence it. I need enough of this region to sequence. If I don’t have enough I won’t get sequence information. So I use PCR to amplify it to a level, and now I can sequence it. What am I going to see? I’ve got two copies of this gene, one of which is a mutant copy, one of which is not. So when I amplify this little region of the gene, what will I sequence? Well, I’ll sequence both copies of what I’ve amplified. So I’ve got a wild type sequence and a mutant sequence, and I can tease these apart.

We also use PCR to look at [RNA] transcripts. There are multiple transcripts; there are millions of different transcripts in each cell. I would want to analyse and quantitate the level of a particular transcript. So I’d need to pick that transcript up from the millions of others, and try to quantitate that, and I am using PCR for that as well. I would isolate the RNA, reverse transcribe it to DNA, and then amplify that.