Frequently Asked Questions

Q: What is the difference between Klentaq and full-length Taq?

A: Klentaq is a truncated version of Taq, which lacks the 5’>3’-exonuclease domain of the enzyme. Klentaq has improved fidelity and is more thermostable and robust than Taq. The fidelity of Omni Klentaq vs. Klentaq is not compromised. Cesium Klentaq has slightly better fidelity than Klentaq. The fidelity of the rest of the mutant enzymes has not been fully determined yet.

The sensitivity of our mutant enzymes is not compromised at the expense of their special inhibition-resistance or cold-sensitivity features, when compared to top commercial Taqs, and allow single gene copy detection from human DNA. The Klentaq enzymes typically require longer than Taq extension times for high yield amplification, and we recommend 3-4 minute extension per 1 kb DNA target.

Q:  What is the advantage of your LA enzyme versions?

A: The “LA” stands for “long and accurate”. The LA enzymes are blended with a proofreader, and perform with better fidelity.  Also, they are the enzymes of choice for long DNA targets, with which they perform exceptionally well. In addition, they are generally more robust, therefore short targets amplification can also benefit from them.

*TIPS on long targets:  For purified DNA templates, consider enzyme concentration of at least 0.1 ul/1 kb target/ 50-100 ul reaction volume, and allow 3-4 min extension time per 1 kb target.  For crude samples, more enzyme may be needed, depending on the level of inhibition (enzyme titration is strongly recommended). Consider including our PEC enhancers.

Q:  What are the practical advantages of your inhibition-resistant Taq enzymes and special PCR enhancers?

A: Our novel enzymes are mutants of Taq DNA polymerase, specially selected for their high resistance to widely encountered potent PCR inhibitors, such as whole blood, serum, plasma, urine, humic acid, bile salts, plant tissues extracts, various food related inhibitors, GITC, ethanol  and others.  As a result, these enzymes can tolerate such inhibitors in PCR at levels where most commercial Taq products fail to perform. This unique feature of the Taq mutants allow for direct PCR of various clinical, environmental, and forensic crude samples, skipping the DNA extraction steps.  Our PCR Enhancer Cocktails (PECs), specially formulated for most inhibitory crude PCR samples, can further raise the tolerance of reaction to inhibitors. In addition, some of our PECs are designed to help also with difficult, GC-rich targets. Due to the above mentioned qualities, our novel PCR enzymes and enhancers should certainly reduce the labor time, cost, and false negative results of your challenging PCR.

Q:  Which enzyme and/or enhancer would work best for my PCR?

A: Our unique selection of mutant Taq enzymes, rendered tolerant to various potent PCR inhibitors, along with our PCR enhancers, enables a variety of direct PCR applications, skipping in many cases DNA extraction prior to PCR. The choice of enzyme / enhancer depends on the type and concentration of the inhibitory substance, the type of qPCR assay, hot-start PCR dependence, GC-content of the template, and other factors. Please refer to Tables “Which Enzyme is Best for Me” and “Which Enhancer is Best for Me”.

Q:  Are the Taq mutant enzymes suitable for real-time PCR?

A: All of our enzymes work quite well with intercalation dyes, like SYBR Green.  Actually, they possess 2-3 times higher than wild-type Taq resistance to SYBR Green (this dye is inhibitory to PCR for plain Taq at more than 1X concentration), which gives them certain advantage in qPCR, especially in reactions with some fluorescent quenching.

For TaqMan assay we only recommend our full-length Taq mutants (the OmniTaq series or CesiumTaq), but not the Klentaq ones, as they lack the 5’>3’- exonuclease activity, required for cleaving the TaqMan probe.

Tip: For tough / inhibitory qPCR samples, 1:1 blends of full-length Taq and Klentaq enzymes may work better than the full-length enzyme alone. (The Klentaqs are generally more robust, and a half amount of the full-length Taq enzyme is sufficient for probe cleavage). If you would like to try this, the two buffers should be blended in the same proportion as well.

Note: The LA versions of our enzymes are not optimal for TaqMan assay, due to the proofreader activity in them.

Q:  I want to amplify DNA targets directly from liquid blood or blood FTA cards, without DNA extraction, what product should I select, and do I need to modify my cycling conditions?

A: We suggest that you select Omni Klentaq, Omni Klentaq 2, OmniTaq or OmniTaq 2, combined, if necessary, with PEC. We strongly recommend enzyme titration for such crude samples, as more enzyme helps with more blood.  Please keep in mind that the optimal amount of enzyme for samples with blood would probably be far in excess of the optimal amount of enzyme for purified DNA samples, an excess enzyme may result in over amplification. Usually 5-10 times less enzyme is recommended with purified DNA.  If even higher amount of the enzyme (at least 1 ul/25 ul reaction) is not satisfactory, you can include PEC enhancer to further boost the blood resistance. We do not recommend using PEC for your purified DNA unless the target is GC-rich and tough. 

You may also use our 5X PCR kits formulated with these enzymes (which provides convenience, but limits the option to titrate the enzyme).

As per the cycling conditions, we suggest that you use your target-optimized PCR protocol with two changes:  A. Introduce a longer initial heating step of 8-10 min at 94-95 deg. prior to cycling, and B. double or triple your extension time.  Please note: If you use a PEC enhancer, keep in mind that it may reduce your optimal annealing temperature by several degrees.

Note: The same considerations apply for other inhibitory crude samples after selecting the right enzyme for your application.

Q:  Besides conventional, or end-point PCR, can I perform qPCR in whole blood with the inhibition-resistant Taq mutants?

A: Yes, with some easy changes in the protocol. Our novel enzymes can resist as high as 40% blood in PCR. Therefore, in qPCR of blood, the amplification should be OK (and you should see your expected products if you want to run them in agarose gel). However, a complication with the optical detection of the amplified products in qPCR stems from the fluorescence quenching effect of the heme. If you use SYBR Green, a remedy to this problem is to simply raise the input dye concentration to 20-30 X (for 5-10% blood), or even higher, depending of the blood concentration. This will compensate for the quenching effect, and will reduce the background.

If you use TaqMan assay, again due to the quenching effect of blood, we recommend raising the fluorescent probe concentration to 400-500 nM, and not exceeding 4-5% blood.

Note: these protocol changes are not necessary if you amplify serum, plasma, or other non-quenching crude samples.

Q:  I obtain negative results in PCR even after DNA purification, what is the reason and how to solve this problem?

A: Some DNA extraction kits cannot completely remove PCR inhibitors from the starting material, such as blood or plant tissue components or humic acids, and can even introduce some inhibitors, such as traces of phenol, chlorophorm,  guanidinium thiocyanate or ethanol. This problem may be eliminated by using our inhibitor-resistant mutants of Taq after a careful titration of the enzyme. (This should be a relatively “easy job” for our enzymes as compared to crude PCR samples, and you’ll have to make sure you don’t overdose the enzyme).

Q:  What is difference between your Cold-Sensitive Taq mutants and other hot-start enzymes?

A: Our cold-sensitive enzymes, such as Cesium Taq, Cesium Klentaq AC, and Cesium Klentaq C, display a suppressed activity at low temperatures, while they become fully active above 65 degrees, thus performing with an intrinsic hot-start in PCR. Unlike hot-start Taq formulations, involving anti-Taq antibodies or chemical modification of the enzyme, they do not require any changes in the cycling conditions.

Q:  Why are the enzymes offered in a volume / reactions format, instead of Units concentrations?

A: The enzyme Units concentration provided by some Taq manufacturers is determined by a slightly modified version of the conventional DNA polymerase activity assay, developed earlier for non-thermostable DNA polymerases. This assay, based on a relatively simple and “easy” reaction, only measures the nucleotide incorporation into nicked DNA at 72 degrees, and does not account for important enzyme qualities in PCR conditions, such as thermostability and processivity. Thus, it is not a per se PCR assay, and does not deliver actual “PCR units”. As a result, it may not manifest and predict the real enzyme performance in PCR. Therefore, we, like other manufacturers, believe that providing a recommended amount of enzyme per reaction is more reliable and practical for the final user. Our suggested enzyme volumes per reaction are based on a series of enzyme titration assays with various target types and sizes.

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 flourescent 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 flourescent 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.