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    KlenTherm™ DNA Polymerase

Products

Cat #

Pack Size  

Price (GBP)  

Qtty

 KlenTherm™ DNA Polymerase

 GC-001-0100  

 100 u

 12.5 

 KlenTherm™ DNA Polymerase

 GC-001-0250  

 250 u

 31.25 

 KlenTherm™ DNA Polymerase

 GC-001-0500  

 500 u

 62.5 

 KlenTherm™ DNA Polymerase

 GC-001-1000  

 1000 u

 125 

 KlenTherm™ DNA Polymerase

 GC-021-5000  

 5000 u

 625 



  DESCRIPTION
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.

  BESCHREIBUNG
KlenTherm™ DNA polymerase ist eine thermostabile Polymerase, welche der von W. M. Barnes beschriebenen KlenTaq Polymerase entspricht. Bei der Expression des Genkonstrukts in E.coli beginnt die Translation erst bei Met236, wodurch die 5’-3’ Exonucleasedomane am N-Terminus wegfallt. Diese Deletion fuhrt zu erhohter Genauigkeit und Aktivitat. Ausserdem macht sie die Polymerase hitzestabiler.
Wiederholtes Erhitzen auf 98°C im entsprechenden Reaktionspuffer beeintrechtigt nicht die Aktivitat! Selbst nach Erhitzung auf 99°C behalt KlenTherm ihre Aktivitat! Solch eine Hitzebestandigkeit besitzt normale Taq Polymerase nicht.
Dadurch ist sie auch fur die thermale DNA Sequenzierung eine hervorragende Alternative zu modifizierten T7 DNA Polymerasen. Besonders problematische DNA Templates mit Sekundarstrukturen und hohen GC-Gehal-ten konnen mit KlenTherm™ DNA Polymerase bei 70°C optimal sequenziert werden. Weiterhin ist die KlenTherm™ DNA Polymerase in Kombination mit einer thermostabilen „proof-reading“-DNA Polymerase (AccuTherm™ ) fur die Amplifikation von besonders langen DNA Fragmenten bis 35 kb geeignet.
GeneCraft bietet solche Mischungen unter dem Namen Synergy™ an.
In bestimmten Applikationen kann KlenTherm™ DNA Polymerase eine optimalere Spezifitat als herkommliche Taq Polymerasen aufweisen, was zur Minimierung von unspezifischen DNA-Amplifikationsprodukten beitragen kann. Gerade bei kurzen Fragmenten genomischer DNA ist KlenTherm™ DNA Polymerase sehr zu empfehlen.
KlenTherm™ DNA polymerase ist vergleichbar mit Taq (USB) und Stoffel fragment (Cetus). Wir empfehlen, bei Fragmenten uber 500 bp mehr Units von KlenTherm™ DNA Polymerase einzusetzen als bei gewohnlicher Taq Polymerase. Aus diesem Grund liefern wir Ihnen KlenTherm™ DNA polymerase in einer Konzentration von 10 u/µl.
KlenTherm™ DNA Polymerase wird mit einem 10x Reaktionspuffer (auf Wunsch auch mit MgCl2 als separater Losung) ohne Aufpreis geliefert. KlenTherm zeigt eine sehr niedrige Aktivität 3´-A-Überhänge anzuhängen.

  APPLICATION

  • 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).


  CONCENTRATION
10 units/µl

  UNIT DEFINITION
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.

  STORAGE BUFFER
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)

  STORAGE TEMPERATURE
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

  COMPANION PRODUCTS
KlenThermN™ DNA polymerase, KlenThermase™, KlenThermaseN™, Synergy™ DNA polymerase, SynergyN™ DNA polymerase, SynergyT™ DNA polymerase, dNTPs

  AMPLIFICATION CONDITIONS

  • 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

  • 58şC 30 sec
  • 72şC 2 min
  • 93şC 20 sec
30 cycles
Fragment is about 1.5 kb

  REFERENCES
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
PURITY OF THE KlenTherm FRAGMENT AND FULL-LENGTH BioTherm

  




 
 
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