KlenTherm™ DNA polymerase is thermostable polymerase corresponding to the KlenTaq polymerase described by W. M. Barnes. It is a N-terminally truncated Taq DNA polymerase. As expressed from a gene construct in E.coli, translation initiates at Met236, bypassing the 5'-3' exonuclease domain of the DNA polymerase-encoding gene. This deletion leaves a highly active and even more heat-stable DNA polymerase activity. Repeated exposure to 98.C, in the recommended reaction buffer, does not seem to diminish the enzyme activity. Signi-ficant activity remains even after exposure to 99.C. The full length enzyme does not tolerate these treatments. Therefore KlenTherm™ DNA polymerase is an excellent alternative to modified T7 RNA polymerase in thermal sequencing methods. Even problematic DNA templates with secondary structures and GC-rich regions can be sequenced at 70°C.
You can use KlenTherm™ DNA polymerase also for Long-PCR up to 35 kb in combination with thermostable „proof-reading“ polymerases (e.g. AccuTherm™ ). GeneCraft offers several mixtures of KlenTherm™ DNA poly-merase called Synergy™.
In special applications KlenTherm™ DNA polymerase has proven better specificity than regular Taq polymerase. This results in minimising of unspecific DNA amplification products.
KlenTherm™ DNA polymerase is similar to, yet disitinct from, USB Taq and Cetus Stoffel fragment. You will need more KlenTherm than Taq protein if the nucleic acid incorporation is more than 500 bp. KlenTherm™ DNA polymerase is shipped at higher (10 u/µl) concentration, so that it can easily incorporate 2 kb, if the same quantity is used as for full-length Taq. The use of KlenTherm™ is espacially recomended for amplifications of small fragments from genomic DNA.
The 10x reaction buffer (on request with or without MgCl2) is delivered free of charge. KlenTherm has a very low 3´-A-Overhang-adding activity
- Fidelity The relative mutation rate during polymerization is twofold lower for KlenTherm as compared to the full-length Taq DNA polymerase.
- Cycle sequencing The absence of the 5'-3' exonuclease activity makes KlenTermTM especially suitable for cycle sequencing. It gives higher sequence intensity and very low backgrounds.
- Long PCR KlenThermTM in combination with a Pfu DNA polymerase (AccuThermTM) exhibiting a proof-reading activity can amplify up to 35 kb DNA fragments.
- Mutation analysis KlenThermTM has a reduced tendency to extend a mismatched 3'-oligonucleotide end making it suitable for mutation analysis with mutationspecific oligos (ARMS analysis).
One unit is defined as the amount of enzyme that incorporates 10 nmoles of dNTPs into acidinsoluble form in 30 min at 72.C under the assay conditions 25 mM TAPS (tris-(hydroxy-methyl)-methyl-amino-propanesul-fonic acid, sodium salt) pH 9.3 (at 25.C), 50 mM KCl, 2 mM MgCl2, 1 mM .-mercaptoethanol) and activated calf thymus DNA as substrate.
10 mM K-phosphate buffer pH 7,0; 100 mM NaCl; 0,5 mM EDTA; 1 mM DTT; 0,01% Tween 20; 50% glycerol (v/v)
Store KlenTherm™ DNA polymerase below 0°C, preferably at -20°C, in a constant temperature freezer.
10X REACTION BUFER
500 mM KCl, 100 mM Tris-HCl (pH 9 at 25°C), 1% Triton X100
Extra solution: 50 mM MgCl2 , add MgCl 2 to a final concentration of 3.5 mM.
Please note the difference between KlenTherm™ and BioTherm™ reaction buffers!
1.5 ml 10x reaction buffer Cat. No GC-001-006
KlenThermN™ DNA polymerase, KlenThermase™, KlenThermaseN™, Synergy™ DNA polymerase, SynergyN™ DNA polymerase, SynergyT™ DNA polymerase, dNTPs
- 10x reaction buffer 3 µl
- 50 mM MgCl22.1 µl
- dNTP Mix10 (end concentration 200 µM) 0.6 µl
- human genomic DNA (300-600 ng) 1 µl
- forward primer (25 pM) 2 µl
- reverse primer (25 pM) 2 µl
- KlenThermTM (10 units/µl) 0.5 µl
- H2O 18.8 µl
total 30 µl
- 58ºC 30 sec
- 72ºC 2 min
- 93ºC 20 sec
Fragment is about 1.5 kb
1 Barnes W.M. 1992. The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. Gene 112: 29-35
„PCR conditions efficient enough to allow the amplification of a 5-kb fragment result in less than half asmany mutations, when KlenTaq is the DNA polymerase, compared to AmpliTaq.“
2 Barnes W.M. 1994. PCR amplification of up to 35-kb DNA with high fidelity and high yield from bacteriophage templates. Proc. Natl. Acad. Sci. USA 91: 2216-2220
„A target length limitation to PCR amplification of DNA has been idientified and addressed. Concomitant-ly, the base-pair fidelity, the ability to use PCR products as primers and the maximum yield of target frag-ment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of DNA template.
Amplification of DNA spans by the polymerase chain reaction (PCR) has become an important and wide-spread tool of genetic analysis since the introduction of thermostabile Taq (Thermus aquaticus) DNA poly-merase for its catalysis. Two limitations to the method are the fidelity of the final product and the size of the product span, that can be amplified. The fidelity problem has been partially addressed by the replace-ment of Taq DNA polymerase by Pfu (Pyrococcus furiosus) DNA polymerase, which exhibits an integral 3'-( editing)-exonuclease, that apparently reduces the mutations per base per cycle from about 10-4 to about 10-5. It was found, that this enzyme is unable to amplify certain DNA sequences in the size range of 1.5 - 2 kb, that Klentaq1 (N-terminal deletion mutant of Taq DNA polymerase analogous to the Klenow fragment of Escherichia coli DNA polymerase I) or AmpliTaq (fullength Taq DNA polymerase) can amplify handily, and Pfu is no more able (i.e., is not able) to amplify DNA product spans in excess of 5-7 kb than is any form of Taq DNA polymerase. For full-length Taq DNA Polymerase and its N-terminally truncated variants Klentaq1, Klentaq5 and Stoffel fragment, PCR amplification rapidly becomes inefficient or nonexistent as the length 3.75U of the target span exceeds 5-6 kb. This is true even if 30 min (10 times longer than seemingly necessary) is used during the extension step of each cycle. Although there are several reports of inefficient but detec-table amplification at 9- to 10-kb target length and one at 15 kb, most general applications are limited to 5 kb. Apparently something has been blocking extension to longer lengths.“
3 Newton C.R. et al. 1989. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucl. Acids. Res. 17:2503-2516.
„The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3' -residue will no function as primers in the PCR under appropriate conditions.“
PURITY OF THE KlenTherm FRAGMENT AND FULL-LENGTH BioTherm