TaKaRa LA (Long and accurate) Taq™ is a thermostable DNA polymerase having 3'-5' exonuclease activity (proof reading activity) that is capable of, under general PCR conditions, higher efficiency and higher fidelity compared to conventional Taq DNA polymerases. This enzyme is basically applicable to any template DNA and especially effective in amplifying more than 15 kb fragments, fragments with high GC content or very repetitive GC content.
What is very unique about this system compared to other polymerases is the use of Takara's GC Buffers (GC Buffer I or GC Buffer II). Unfortunately, Takara does not tell us exactly what is in the buffers. When the GC Buffers are used, amplification of templates having complex secondary structure or high GC content works in comparison to other polymerases which sometimes yield an inefficient amount of product. You can also buy the LA Taq¢â with a traditional buffer for amplification. I do not have experience with the regular buffer.
The reason I used the LA Taq™ with GC Buffers is because I was attempting to subclone two large fragments into different vectors for expression with different tags. I was having difficulty amplifying a 4.5 kb PCR product using Invitrogen¡¯s Platinum PCR Super Mix High Fidelity or Stratagene's Pfu Turbo. My template was a purified plasmid and I knew the plasmid to be correct yet I was not getting PCR product. I tried spiking with DMSO (per recommendations from tech services of each company) and still was getting no yield. After determining the GC content was about 65% and highly repetitive, I realized that the enzyme was having difficulty. After doing some searches, I found this enzyme and buffer components. I set up a standard PCR using both buffers and found that I got robust amplification either the GC Buffer I or GC Buffer II.
The company recommends trying both buffers for initial experiments. In some instances, only one of the buffers will work in amplification while the other one will not. The buffers come supplied as 2X GC Buffer I and II. Because of the proofreading activity, my products had no mutations based upon sequencing analysis.
Typical 3-step cycling conditions can be used for PCR. However, the manufacturer's protocol actually recommends a 2-step PCR with annealing of primer and elongation occurring at 68ºC. I do not use this and prefer the traditional method of a 3-step PCR with different primer annealing and extension temperatures.
In conclusion, I have found this to be an excellent alternative for amplifying fragments rich in GC content where other enzymes appear not to work even in the presence of DMSO or other agents designed to aid in melting of the template.
| Comment on this Review |
Comments? Questions? Discuss this review of LA-Taq-With-GC-Buffer-From-Takara with the author of this review, Brandon White, and the rest of the Biocompare community.
|
Brandon White
Assistant Professor
Department of Biological Sciences
San Jose State University
United States
more articles...
