Mutagenic PCR unsuccessful :( - pls help.... (Nov/16/2005 )

Hi, everybody:

I performed mutagenic PCR reactions two weeks ago, using a 50-base long primer containing two mismatches in the middle. I used the stratagene reaction buffer for these reactions. Because of the insufficient amount of Pfu Turbo left in the site-directed mutagenesis kit, I used the Taq High-fedelity DNA polymerase (which also has proof-reading capability) intead.

When I checked the DpnI-digested PCR products on a 1% agarose gel, I did see some bands which were of the correct size on it. I thought that must be the mutated plasmid DNA, so I did transformation of the mutated plasmid into SCS1 competent cells. Fortunately, I got some colonies which was used in my mini prep experiments.

Last week, I sent the plasmids for sequencing, but found the DNA was not mutated at all.

I really don't know what is wrong with my mutagenic PCR?? Must I use the stratagene Pfu Turbo DNA polymerase?? Any ideas??

thanks,

Recently, I've had to do more and more of this. We found that there was some difference between high fidelity polymerases. Even Pfx didn't give us the same results as Pfu. So now the lab only uses Pfu for mutagenesis. We also use the quick solution with Stratagene's multi kit. With all of this said Stratgene also gives some pretty strict guidelines for successful primers.

25-45 mer
Tm >= 78
optimally the primers should be 40% GC
all of this is from their manual

Good luck and I hope this helps. We have instances where we have had to run the reaction a couple of times. It is pretty sensitive.

Or you could try this: http://nar.oxfordjournals.org/cgi/content/full/32/21/e174

It worked for me last week.

-Matt

hi, i have a little experience with this, but I was fortunate enough to have good restriction sites near the bases I wanted to mutate, so you may not be able to do this...

I PCR amplified a 100bp region containing 2 "good" RE binding sites with the mutagenic primers (the 5' RE was included in the primer sequence, primers were 20bp long) Then clone into pGEM T-easy and subclone back into the vector of interest... I was also able to digest the ligation reaction with enzymes that cut T-easy and not my vector, reducing the number of false positives... Of course you still have to sequence, In my case (this may be the only project I have ever been lucky on ) I sequenced two colonies from each and they all contained the mutated insert...

I used rTaq from Takara for this, perhaps b/c of the short sequence it was not as important to have high fidelity??

Anyway, just another way to approach....

Hope this helps, and good luck!!

The Quikchange method depends critically on the enzyme not exhibiting strand displacement activity. Pfu turbo and Pfu ultra do not strand displace, while all mixtures containing Taq do. You won't get the Quikchange method to work with Taq containing enzymes.

Take a look at the manual of quickchange (you can find it on www.stratagene.com).

Basically, when you do mutagenesis according to the quickchange method, you use 2 primers the opposite direction containing your mutation. This leads to amplification of your entire plasmid. Once your polymerase has reached "the end of the plasmid", it meets up with the primer it was elongating. Polymerases without strand displacement stop here, the primer stands in the way. Polymerases with strand displacement will "eat away" primer, so that you amplify your original DNA (not containing the mutation).

Btw: Pfu's will strand displace @ 72°C, but not @ 68!

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 have 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 have shortcomings 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). Ko’s method has been very successful for every situations mentioned above.

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: Ko’s Type IIs method!
* Easy way but need money: company rather than kit!

For more discussion, you can contact to choik1@umdnj.edu