final PCR product - need clarifications (Jan/10/2011 )
I have a few PCR questions..
1. I have a target region and it is surrounded by genomic DNA. I have primers specific for that target region only, so the primers bind to the first few bases of the target region on either side and amplify only that region out, I amplify with PCR for 30 cycles and I get my target region. But can someone confirm this isn't the best way to go about it, rather it is better to have the primers bind a few nucleotides outside the target region rather than directly on the target region itself to ensure all the target region will get amplified? But does this mean the final PCR product contains the target region flanked with those few unnecessary nucleotides amplified along with the target region?
2. Say one of my primers contains a barcode so I want the PCR product to be target region plus barcode. But how does the barcode get into the final PCR product? How does this PCR work? Wouldn't this mean that #1 would also have some amount of primer sequence in its final PCR product too?
I appreciate any help
1. It depends a bit on the context. If you have e.g. a variable DNA region as target (i.e. with high mutation rate), it is useful to have the primers in the flanking region, which is more or less highly conserved. Otherwise your PCR won't work or only in a few of the samples/organisms. Then you have of course the primer binding region too in your PCR product, but as it is everywhere the same, it doesn't matter (if you look at the size of the products, or if you sequence it later).
2. I know barcodes only figuratively as DNA region that has an unique sequence and therefore identifies e.g. a species similar to a printed barcode that identifies a consumer good. If you need this to introduce to the product, you can also add such a sequence to a primer normally to the 5' end that hasn't to bind to the template. It's like a tail then.