FAQ sheet for Wayne Barnes' thermostable enzymes
[Actually, this sheet only has the answers, not the questions.]
Klentaq1 is a Klenow-fragment analog of Taq DNA polymerase. It is the most thermostable form of Taq known, it has no exonuclease or endonuclease whatever, and its crystal structure is known [4].
LA PCR is Long and Accurate PCR [1].
The suffix LA means a minor proofreading enzyme has been mixed in, according to U.S. Patent 5,436,149.
KlentaqLA (KTLA) or TaqLA or TthLA are the best enzymes for long DNA extensions at high temperature, such as primer-directed mutagenesis, long PCR or high fidelity PCR, or priming with PCR products (megaprimers). (These "enzymes" are actually mixtures, U.S. Patent 5,436,149, invented by Wayne Barnes, and now owned by Takara BIO, Inc., Japan).
- Watch this space for my improvements on the fidelity.
- TaqLA is sold by Boehringer-Mannheim as "Expand", by Takara Shuzo as "ExTaq for LA PCR", and by Life Technologies as "Elongase".
- TthLA is sold by Perkin Elmer as "Tth XL" and by Clontech as "Advantage Tth" - "TaqPlus" and "Taq Extender" from Stratagene also apply U.S. Patent 5,436,149.
"LA Technology" is the concept of mixing two thermostable enzymes together to get longer and more accurate (higher fidelity) DNA extension, usually for PCR. Invented by Wayne M. Barnes, U.S. Patent 5,436,149 and foreign counterparts. The patent is now owned by Takara Shuzo (contact for licenses: Mr. Junichi Mineno, fax 011-81 775-43-2312.)
Both of the following recommended buffers assume that you are adding dNTPs with their own (equimolar) magnesium. If you are adding 250 uM each dNTP, add therefore an extra 1 mM magnesium (final 3.5 mM) to achieve this.
We do this by having a stock of 10 mM each dNTP, 40 mM MgCl2 ("10/40"). Then, the following buffers will supply the optimal "excess" magnesium when they are at 1X. [ You may prefer to add an additional 10 mM MgCl2 to the following, and then add dNTPs with no Mg.]
The Tris-HCl stock is made from Trizma Base and HCl, at 1 M Tris. The pH is however read at 50 mM in otherwise plain water at room temperature.
* 10xKLA for Klentaq1, KlentaqLA, and Taquenase is
* 500 mM Tris-HCl pH 9.2; 160 mM ammonium sulfate; 25 mM MgCl2; 1% Tween 20.
* 10xTLA for Taq and TaqLA is the same, but only 7.5 mM MgCl2.
* Include final 1.3 M betaine for high GC targets and for multiplex PCR. Also reduce heat step to 92-93 deg. C. [modified from ref. 2 & 5]
Our 'bench-strength' Klentaq1, KTLA, and TAQUENASE are equivalent to the activity of wt Taq if they are used for an amplification of 2 kb. That is, 0.1 ul is just right for a reaction size of 50 ul. Other vendors of (wild-type) Taq call this 5 units/ul; we call it '5 2kb-PCR-units'/ul. More Klentaq1 is needed for longer targets, up to a maximum of 0.65 ul/50 ul (target size of 35 kb; requires KTLA instead of Klentaq1). Less Klentaq1 is needed (but 0.1 ul is still okay) for targets smaller than 2 kb.
For PCR targets under 2 kb, 1/2 as much enzyme (TaqLA or KlentaqLA) is needed if the extension temperature is lowered to 60 deg. and the extension time is increased somewhat (i.e. from 5' to 8' or 10').
Klentaq1 is good for short PCR, RAPDs, etc. For cycle sequencing, it is excellent, but it is now old tech compared to Taquenase.
Does KlentaqLA leave a blunt end on its PCR products? Partly yes and partly no. Klentaq1 does put the extra A on. To do this efficiently, however, takes an extra long last extension (20-30 minutes). The proofreading
enzyme in the mixture is expected to remove this extra A. Which one wins may depend on the exact treatment your PCR reaction got after it finished
cycling. Possibilities are:
a. Overnight at 4 degrees.
b. Over lunch at 25 degrees.
c. Over coffee at 68 degrees.
d. Any of the above, followed by a phone call at 25 degrees.
e. Other than the above.
- Have I done the experimental conditions above and then examined the ends
of the DNA? No.
- Have I cloned my products into blunt-ended vectors? Yes, with 1 mM hexammine-cobalt chloride and 1 mM DTT during the blunt ligation by T4 DNA polymerase.
- Have I checked the reading frame across the cloning site? Not by sequencing.
- Have I ever cloned a reading-frame-critical PCR product made by KlentaqLA into a blunt site in an ORF? Yes, and about half of the clones seem OK by enzyme activity of the encoded product.
- Have I ever used a T-vector? No.
We have data that Taquenase is the best enzyme for cycle sequencing. It must be used with MnSO4 (in addition to MgCl2) to get the best results (Barnes, unpublished). Unfortunately, due to patent and licensing problems (see below), you can't use it.
1.25 M betaine (Sigma no. B-2629) is also a good idea to include in cycle-sequencing reactions [Barnes, unpublished]
"Taquenase"(tm) is a combination of two mutants of Taq. Mutant 1 is an N-terminal deletion Klentaq1 (U.S. Patent 5,436,149 and other country patents pending. Mutant 2 is F667Y discovered by Stan Tabor [3]. The F667Y mutations allows 100 to 1000 fold less ddNTP to work well. As of March 25, 1997, this mutation is covered by US patent 5,614,365 issued to Tabor & Richardson and licensed exclusively to Amersham. Therefore we can not provide this enzyme unless we get a license from Amersham, which is not expected.
We believe there is no patent covering cycle sequencing (invented by M. Craxton, MRC Cambridge, England), nor dideoxy sequencing (invented by Fred Sanger, MRC Cambridge, England).
ddNTP means "dideoxy NTPs" or "dideoxy terminators".
NTP means nucleoside triphosphate, the building block for polymerase. They can be ATP, GTP, CTP, or UTP==TTP. For RNA, sometimes the prefix r is added for clarity, such as rCTP. For DNA, usually the prefix d is added, such as dATP.
Dideoxy means two positions with no OH group (the 2' and 3' positions).
Perkin Elmer has recently raised the price (by reducing the concentration) of their fluorescent ddNTPs by 1000-fold .
DYE-ddNTP means "DYE terminators" or DYE-dideoxy terminators, which is getting to be a mouthful. They are sold by ABI, but they were originally invented by Dupont, and are available from their subsidiary N.E.N., or will be soon.
DYE means a fluorescent label, activated by laser beam on the sequencer machine.
Klentaq1 is the most stable form of Taq DNA polymerase known, as tested by PCR at with heat steps at 98 or 99 degrees. Taquenase retains this heat stability. Since Thermo Sequenase is Taquenase with 7 additional amino acids, we believe it has this increased thermostability, also.
References:
[1] Barnes, W.M. (1995) U.S. Patent No. 5,436,149. Thermostable DNA polymerase with enhanced thermostability and enhanced length and efficiency of primer extension.
[2] Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A., & Weissman, S.M. (1996) Uniform amplification of a mixture of deoxyyribonucleic acids with varying GC content, Genome Research 6:633-638.
[3] Tabor, S. & Richardson, C.C. (1995) A single residue in DNA polymerases of the Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides, Proc. Natl. Acad. Sci. 92:6339-6343.
[4] Korolev, S., Nayal, M., Barnes, W.M., DiCera, E., & Waksman, G. (1995), Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5 A resolution: Structural basis for thermostability, Proc. Natl. Acad. Sci. 92:9264-9268.
[5] Weissensteiner, T. & Lancchbury, J.S. (1996) Strategy for controlling preferential amplifications and avoiding false negatives in PCR typing, BioTechniques 21:1102-1108.
Wayne M. Barnes, Ph.D. wayne@barnes1.wustl.edu
DNA Polymerase Facility or
Biochemistry Dept. 8231 barnes@biodec.wustl.edu
Washington Univ. Medical School 314.362.3351 fax 7183
http://barnes1.wustl.edu/~wayne
Just plain Taq is old tech anymore.