Site-directed mutagenesis
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Site-directed mutagenesis
I have a cloned gene. I want to mutate a single base-pair. Can anyone recommend any good protocol. Which kit do you suggest?
Malay
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I 'd save your bucks and use the overlap extension method. It's fast, efficient and cheap (in your case, only need 2 mutagenesis primers + your 5' and 3' primer for the gene, which you should have already).
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I agree with this, PCR overlap mutagenesis is MUCH better than most methods. More efficient, easier to do, just need to clone the fragment back into your vector and that's it!
Kevin
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Last update 24-Sep-2001, Rating Good of 3 votes.
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does it matter if we use PAGE purified primers
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For rapid and cost-effective method, I strongly recommend recently published method using type IIs enzyme (Ko, JK and Ma J, Am J Physiol Cell Physiol. 2005, 288: C1273-8.). This method originally published by Tomic M (Nucleic Acid Res. 1990, 18: 1656.). My colleagues and I also successfully generated various mutants (substitution, insertion, deletion and chimeragenesis) by this method. You need only four primers (just desalted not purified, it’s very cheap!) and type IIs restriction enzyme. The mutagenesis efficiency is close to 100%.
Presently, the overlap extension method, megaprimer method, Quick Change method (Stratagene) are prevalent among numerous PCR-based mutagenesis methods developed commercially or non-commercially. However, all of them has shortcoming for applying to the diverse mutagenesis strategies including base substitution, deletion, insertion, chimeric gene generation and multiple-site mutagenesis. Especially, they show low mutagenesis efficiency (below 40%) for genes with long sequence (>3 kb), GC-rich region, tight secondary structure, or tandem repeats and for a long frame mutation (> 10 bp).
If you have plan to use commercial kits, another choice for short way for your research is use of mutagenesis service company. I realized the mutagenesis cost using commercial kit is never cheap! My estimate is $200 ~ 250 per one mutagenesis reaction using kit (one kit rexn., $20 + purified two primers, $150 + three clone sequencing, $30 + subcloning reagent, $30 : SM, media, agar plate, buffer, tube, tips --- and time). Besides, purchasing kit for just one or two mutant generation is money wasting because remaining kit reagents are useless. Recent biotech companies provide rapid and precise mutagenesis service in affordable prices. Some people in our lab use Mutagenex Inc. (USA) and I found other companies offering in low price:
Mutagenex: $249 per mutation, USA
Topgenetech: $269 per mutation, Canada
MCLab: $280, USA
You can also find more other companies that have different technology and service criteria.
In conclusion, my recommendation is,
Efficient and cheap method: Type IIs method!
Easy way but need money: company rather than kit!
For more discussion, please contact to choik1@umdnj.edu
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I have exactly same problem-I always got shorter PCR product instead of expected the whole vector. Does anyone know the answer for this? I appreciate for any helpful suggestion.
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i am also having problems with the third PCR using overlapping primers.i am always getting bands corresponding to the positions of individual primers
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Hi
I am trying to find a way to introduce three new amino acids in a recombinant protein, preferrably by PCR. I thought a technique similar to site-directed mut. might work. Does anyone have experience with that?
Rasmus
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I am doing a site-mutation too on a gene construct. so far, i can't seem to amplify the whole vector DNA using two overlapping primers, instead, I got mainly shorter fragments. Can you tell me a little bit in detail how this method works and what kind of Taq will do? thanks.
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Related resource
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