I'm beginning PCR..... - (Jul/16/2008 )
I have some questions about how the PCR process will work...
I am cloning two genes into yeast.
I have the genomic DNA for two archea.
According to my understanding, I design primers that anneal to my gene. PCR will amplify starting at the primers, but what happens to the rest of the genome. If I design restriction sites into my primer, then I cut out the gene after it has been amplified. Does this take care of the excess DNA (the rest of the genome I don't want)? Also, do the restriction enzymes cut through both strands of DNA? Because the final 3' end on my forward primer side won't have a restriction side on that side of the DNA (same with the 3' end on the reverse primer).
Once I've cut out the amplified gene, it is going into a yeast shuttle vector. I assume that a combination of DNA ligase and restriction digests will recombine the gene into the vector, from there, I just have to perform the transformation for the yeast.
Is this how the process will roughly go?
Thanks so much,
I had never thought of it, but here's how I understand it.
The first round of PCR, theorically would never stop, or would stop at the end of the chromosome (or genome, if you use circular DNA). But in PCR, you design your run to have a certain amount of time during witch the polymerase duplicate the DNA. So for the first cycle, the polymerase will stop when the denaturation (step at 95) will occur. Then, the primers will bind to both the incomplete genome and the original genome, and this will continue on. But since you will have more "small" products than full genome, the further the reaction takes place, the more exact amplicon there will be. So you will finally have almost only the desired amplicon.
As for the cut, yes, the restriction enzyme do cut both strands of DNA. And for your 3' strand, remember that the DNA polymerase synthetise DNA from the other strand's matrice. So you restriction site will be incorporated from the other strand.
I have a feeling i'm not clear, answers from others are welcome
yeah.. you're right madrius
I'm not sure if I get what you mean, but try this. You can engineer restriction sites into each primer, and typically they will be two different sites (to give you directionality of the cloning step: I mean you don't want the insert to go in back to front, do you?). When you do the PCR, after the first few rounds of amplification, the majority of DNA present will be your gene, and only your gene. Madrius ssaid that the new strand will keep on being synthesised from genomic DNA, but if the primer binds to a newly-synthesised strand, extension will terminate at the end of the strand. Google PCR theory, and look at the images. It will help you see what is happening. Try http://www.answers.com/topic/polymerase-chain-reaction and scroll down to figure 2.
Once you have done the PCR, and cleaned up the product from everything else, you just do the two RE digests, and you'll end up with the gene of interest flanked by two unique restriction sites. You should remember, however, to select restriction sites that don't appear in your gene, or else you'll just start chopping up your PCR product!!!
See Figure2 in Chapter2 of the book POLYMERASE CHAIN REACTION: THE TECHNIQUE AND ITS APPLICATIONS by Rosalind and Alasdair. Hope the figure explains the answer to your question in simple.