5 replies to this topic

[mcb56]

Hello I have a general question about PCR mutagenesis on a plasmid. I have heard that this is not exponential amplification, it only yields a 1:1 ratio of parent and mutant plasmid. Why is this the case? The primers should anneal to both the parent plasmid and the mutant during each round of amplification.

Thank you

[Ameya P]

mcb56, on 19 September 2010 - 09:57 AM, said:

Hello I have a general question about PCR mutagenesis on a plasmid. I have heard that this is not exponential amplification, it only yields a 1:1 ratio of parent and mutant plasmid. Why is this the case? The primers should anneal to both the parent plasmid and the mutant during each round of amplification.

Thank you

Mcb,

I think the amplification is exponential, as with other PCRs, but the product you are interested in and the parent plasmid have a 1:1 ratio. Since your primers have (some)bases which are not complementary  to the template, the products obtained after the first cycle of PCR (theoretically) have hybrid status consisting of one parent strand and one mutant strand. Its only after the second cycle, that the mutant region of the DNA is incorporated in the DNA and starts getting amplified. In the meantime, your parent DNA has also increased in equal amounts and will continue to multiply in the same manner as your mutant DNA. Thus, the ratio stays 1:1.

Draw a figure and you will realise what's happening....  

[phage434]

This isn't right.  Every copy incorporates a molecule of the primer oligo, and thus has the sequence of the primer as part of the copy. After the first few cycles, the modified plasmid will dominate.  It will also bind the primers better, since it has a perfect match.  The high template concentration and low cycle count are an attempt to reduce mutations at other sites on the plasmid.

[donny]

Hi mcb56,

If you're referring to the Stratagene QuikChange protocol, yes, it is linear amplification (it doesn't yield only 1:1 ratio relative to the parental DNA, though). Due to the primer design in this protocol, the primers cannot use the PCR product as the template because they anneal to the 5' end of the DNA and not the 3' end as in "normal" PCR. Therefore, the only template present is the parental plasmid. I've attached a file that will hopefully help illustrate this. .

However, there are other protocols, such as GeneTailor (Invitrogen) and ExSite (Stratagene), that seems to amplify exponentially to me. The difference between these protocols and QuikChange is that the primers in the latter protocols have sequences overlapping 3' ends of the amplified DNA, hence are able to facilitate amplification of the DNA strands. Check out the user manuals for these kits.

Hope that helps.

Attached Files

•  Quikchange.txt   4.58K   200 downloads

Edited by donny, 20 September 2010 - 06:44 AM.

[samita]

then how it get circularized?????

donny, on 20 September 2010 - 06:40 AM, said:

Hi mcb56,

If you're referring to the Stratagene QuikChange protocol, yes, it is linear amplification (it doesn't yield only 1:1 ratio relative to the parental DNA, though). Due to the primer design in this protocol, the primers cannot use the PCR product as the template because they anneal to the 5' end of the DNA and not the 3' end as in "normal" PCR. Therefore, the only template present is the parental plasmid. I've attached a file that will hopefully help illustrate this. .

However, there are other protocols, such as GeneTailor (Invitrogen) and ExSite (Stratagene), that seems to amplify exponentially to me. The difference between these protocols and QuikChange is that the primers in the latter protocols have sequences overlapping 3' ends of the amplified DNA, hence are able to facilitate amplification of the DNA strands. Check out the user manuals for these kits.

Hope that helps.

[donny]

samita, on 22 September 2010 - 07:53 AM, said:

then how it get circularized?????

The QuikChange and GeneTailor protocol use primers that overlap. Upon denaturation and annealing, they hybridise and the overlapping part of the primers hold the ends of the dsDNA together like an oversized restriction digested "sticky end". The last figure in my attachment from the last post illustrates this. The plasmids then have to be transformed into a bacteria host to seal the nick and replicate the plasmids. ExSite is slightly different. The product has no "sticky ends". So, 5' phosphorylated primer is required so that the products can be circularized by ligation with ligase.