PCR MUTAGENESIS PROBLEM - SiteDirected Mutagenesis - Problem (Jul/21/2005 )
Im doing invitro pcr mutagenesis DpnI digestion method
Primers size is 32 bp. both primers got G or C in terminal ends
Using LA Taq - TaKaRa
After PCR reaction did DpnI digestion and transformed into Ecoli Dh5al
Then all clones having insert and plasmid
Problem in sequence confirmation
Problem is: noticed the mutated region after that that primer sequence it is repeated many times. That was the problem
Can any body help appreciated.
Thanks in advance
How many colonies have you sequenced? You just screen some more colonies and the right one will appear.
I met such problem also when I was using stratagene site-directed mutagenesis kit. I sequenced 3 colonies, only one of them was normal, the rest got repeated primer sequence.
If you're up for it, you can check your primer sequence for complementary (or near complementary) sequences at either end. A student in our lab had this same problem (primer duplication) and it was due to partially overlapping sequence at either end. The slight overlap was allowing aberrent priming during the annealing step, which resulted in an additional copy of the primer in the middle of the construct. If this is happening, your sequence should show incomplete primer sequence repeats (ie, most of the primer shows up as a duplicate, but it's missing 4-10 residues before it starts the next duplicate sequence).
If you observe that, there's a couple things you can try.
1) Shorten the primers to exclude one of the overlapping regions.
2) Redesign the primers with wobble mutations to disrupt binding in the overlapping region.
3) Repeat the PCR with a higher annealing temperature. You won't generate as much product, but you should reduce the less favorable aberrent priming and eliminate sequence duplications.
Hope that helps.
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 firstname.lastname@example.org