I have designed primer for site directed mutagenesis to change only one amino acid in my interest of gene (which is cloned into pBluescript vector) so forward and reverse primer are complementary to each other, except one base pare. For this I have performed site directed mutagenesis PCR reaction independently by using forward primer in one tube and reverse primer in second PCR tube and in third PCR tube I added both forward and reverse primer. After PCR I mixed amplified mutated sample from tube 1 and tube 2 and performed DpnI digestion and I have also DpnI digested amplified sample from PCR tube 3. I wish to understand the reason and molecular reaction going inside the tube for independent PCR reaction for Forward (F) and Reverse(R) primer and PCR reaction for both F+ R primer.
Monika, I would not think that there should be much difference between the two approaches. You are not actually doing a PCR reaction here, but instead a linear chain reaction, where the initial input DNA is primed multiple times to form a linear product that covers the entire plasmid. When the two synthesized strands are put together, they can anneal into a circular DS DNA molecule that is held together by the overlap at the oligonucleotide primers. These circles can transform bacteria, unlike any of the linear products that will be degraded if put into bacteria.
The only real difference here should be that when you have both primers in the same tube, you can have some competitive hybridization of the primers with one another and with the products from the other strand. This might lower yields slightly.
The Dpn treatment just degrades the original plasmid DNA, which could transform bacteria at a very high efficiency if it were left intact.
Hi Monika,
Maybe I misunderstood what you wrote. You write that your primers are complementary to each other except for one base pair. In site directed mutagenesis, your primers must be perfectly complementary to each other, but there are mismatches with your template DNA.
Now about your question. When doing a PCR with both primers, both strands of the template DNA are copied and the amount of DNA increses exponentially as there is more and more DNA to copy. The problem with site directed mutagenesis is that the primers are complementary to each other. So they form primer dimers by annealing to each other instead of annealing to the template DNA. Sometimes the PCR works anyway, but sometimes the target DNA is not copied at all. In this case, another approach is to do separated PCRs: PCR 1 with forward primer and PCR 2 with reverse primer. So the two primers never meet and they can anneal only on the template DNA. But in this case, only one strand of the template DNA is copied in each PCR reaction, which means that the amount of DNA increases only linearly, and not exponentially. So if you proceed by separated PCRs, you need large amounts of template DNA if you whant to recover enouth of your mutated DNA. At the end, when mixing both PCRs, the single strands from PCR 1 anneal with their complementary strands from PCR 2 and vice versa.
Single PCR step is required for site directed mutagenesis. Why are you complicating your experiment? Either call me at 09558880617 or mail me @ [email protected].
Primer would not be exact complimentary to each other if you design primer slightly from different region of gene.
There is a modified method (http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91) proved much more efficient if you are struggling with the quick change. The mutagenesis efficiency of the modified method is increased mainly by changing the primer design and PCR amplification cycles.
I only use the reaction with both plasmids, like your tube 3. I dont understand why to do the separate reactions. Its true that in the latter case you can get mutated DNA products, but not a real amplification, perhaps the DNA yields are too low. This can be comepensated by increasing the template amount, but then the risk of getting non-mutated products increases.
Thanks to all. Emeline Vernhes you are right my primer are exactly complementary to each other. Some what I got my answer from your and Prescott's reply. I just want to know the reason behind separate reaction with F and R Primer and simultaneous reaction with both primers.
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