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Polymerases

Polymerases are enzymes which can be found in all living organisms. They catalyze the synthesis of long chains or polymers of nucleotides, subunits of nucleic acids the building block of DNA. They are used in the multiplication of genetic information (DNA) e.g. during replication, a process prior to cell division. In this way, genetic information is transmitted from generation to generation. Additionally they are crucial for the first steps of protein biosynthesis, the production of proteins within a cell.

Polymerases are used to assemble DNA and RNA molecules and usually work in pairs. They link specific sequences of nucleotides to chains using base-pairing interactions. This sequences is determined by another chain of nucleic acids which is used as template (fig. 1). Depending on type of template and product (RNA or DNA) one can distinguish between following polymerases:


DNA polymerases adds nucleotides to the 3' end of a strand of DNA.
fig 1. DNA polymerases adds nucleotides to the 3' end of a strand of DNA.


Type Template → Product Function
DNA-dependent DNA-Polymerase DNA → DNA Replication
RNA-dependent DNA-Polymerase RNA → DNA Reverse transcription
DNA-dependent RNA-Polymerase DNA → RNA Transcription
RNA-dependent RNA-Polymerase RNA → RNA Replication of some RNA-viruses


Taq polymerase

Taq polymerase is a thermostable DNA polymerase named after the thermophilic bacterium Thermus aquaticus from which it was originally isolated. Its name is often abbreviated to Taq Pol or simply Taq. It is frequently used in the polymerase chain reaction (PCR), a method for greatly amplifying the quantity of short segments of DNA.

T. aquaticus is a bacterium that lives in hot springs and hydrothermal vents, and Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions (high temperature) required during PCR. Therefore, it replaced the DNA polymerase from E. coli originally used in PCR. Taq's optimum temperature for activity is 75–80 °C, and can replicate a 1000 base pair strand of DNA in less than 10 seconds at 72 °C.

A problem which occurs when working with Taqs is the lack of 3' to 5' exonuclease proofreading activity which lowers the replication fidelty by a big margin. Originally its error rate was measured at about 1 in 9,000 nucleotides. In comparison DNA-Polymerase which can be found in the humand body show mistakes about one in every billion base pairs copied. They proofread after copying so that misplaced base pairs can be corrected. This preserves the integrity of the original DNA strand that is passed onto the daughter cells. And lowers the mutation rate.

Taq Polymerases available at genomics-online:

Name Max Target Length (kb) Extension Speed (kb/min)* Fidelity* Proofreading Kits/Mastermix Applications
HGS Diamond Taq Polymerase 2 60x N
  • PCR
  • qPCR
  • multiplex assays
Taq DNA Polymerase Enzyme for PAGE 3 1 to 2 Y ABIN2953670
  • Short fragment PCR
Taq DNA Polymerase Enzyme (1-2 kb/min) 4 1 to 2 1x N ABIN2953669
  • Routine PCR
  • Colony PCR
Taq DNA Polymerase Enzyme (4 kb) 4 up to 6 1x N
  • Routine PCR
  • High throughput PCR
  • Colony PCR
Taq DNA Polymerase Enzyme (6 kb/min) 4 up to 6 1x N
  • Routine PCR
  • High throughput PCR
  • Colony PCR
Taq DNA Polymerase 6 1 1x N ABIN4219185
  • Routine PCR amplification of DNA templates up to 6 kb
  • Suitable for a wide range of PCR assays
  • TA cloning
Taq DNA Polymerase 6 1 1x N ABIN1536552
  • PCR
  • 3’ A-tailing of blunt ends
  • Primer extension
  • DNA sequencing
Taq DNA Polymerase without Mg2+ 6 1 1x N ABIN1536552
  • Routine PCR amplification of DNA templates up to 6 kb
  • Suitable for a wide range of PCR assays
  • TA cloning
Taq DNA Polymerase, concentrated 6 1 1x N ABIN1536552
  • Routine PCR amplification of DNA templates up to 6 kb
  • Suitable for a wide range of PCR assays
  • TA cloning
HotStart DNA Polymerase 6 1 1x N ABIN4219189
  • Assays with prolonged reaction setup or liquid handling
  • Multiplex PCR
  • Specific amplification of difficult templates (i.e. G-C rich)
  • Low copy PCR assays
  • TA Cloning
Precision™ DNA Polymerase 6 1 60x Y ABIN4219162
  • Whole genome sequencing
  • Site-directed mutagenesis
  • Blunt-end cloning
  • Specific amplification of difficult templates (i.e. GC-Rich)
  • Low copy PCR assays
T DNA Polymerase Enzyme 8 1 to 2 18x Y ABIN2953672
  • Complex templates
  • TA cloning
Diamond Taq DNA Polymerase 10 18x N
  • PCR
  • Ta-cloning
Red Diamond Taq DNA Polymerase 10 18x N
  • PCR
  • Ta-cloning
  • PCR for low levels of of bacterial & fungal DNA
SilverStar DNA Polymerase 10 1x N
  • Extremely robust Taq DNA polymerase giving high yield
  • DNA array
  • Ta-cloning
Green Taq DNA Polymerase 10 1 N
  • PCR (For simple templates up to 10 kb; for complex templates up to 3kb)
  • 3’ A-tailing of blunt ends (T/A-cloning)
  • Primer extension
  • DNA labeling reactions
TaqFast DNA Polymerase 12 4 to 6 10x Y ABIN4219168
  • Fast PCR
  • High-throughput PCR
Kodaq DNA Polymerase 12 1 50x Y General
Tissue
Plant
  • Routine PCR amplification
  • PCR success with AT and GC Rich sequences
  • High-throughput PCR
  • RACE
  • NGS Library construction
Taq DNA Polymerase Enzyme (15 kb) 15 1 to 2 18x N
  • Complex templates
  • GC/AT-rich templates
  • Multiplex PCR
  • High yield PCR
HiFi DNA Polymerase Enzyme (High
Fidelity)
15 1 to 2 18x Y genomic DNA
λDNA, cDNA and plasmid DNA
  • Complex templates
  • GC/AT rich templates
  • Long PCR
  • High yield PCR
TopTaq DNA Polymerase Enzyme 15 1 to 2 18x N
  • Complex templates
  • GC/AT-rich templates
  • Multiplex PCR
  • High yield PCR
Bestaq™ DNA Polymerase 15 3 to 4 50x Y Standard
Safe-green
  • High-throughput PCR
  • Robust amplification of AT- and GC-Rich sequences
  • RACE
  • NGS Library construction
Hot Diamond Taq DNA Polymerase 15 60x N
  • improves PCR efficiency and specificity to amplify AT and GC rich regions, long and difficult templates
  • PCR for low levels of of bacterial & fungal DNA
  • isothermal amplification.
Taq DNA Polymerase 20 1 1x N
  • PCR and primer extension reactions at elevated temperatures
Long-Range DNA Polymerase (PCR) 20 3 to 4 1x N
  • Long range PCR
  • Robust amplification of AT- and GC-Rich sequences
KD Plus DNA Polymerase Enzyme 15 (Plasmid: 20) 1 108x
  • Fast, high specificity amplification
  • High fidelity, high yield amplification

Pfu Polymerase

Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus. Pfu DNA polymerase has superior thermostability and proofreading properties compared to Taq DNA polymerase. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand. Consequently, Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts.

Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6% mutated products when amplifying 1 kb fragments using PCR. Therefore it is useful in PCR reactions which need sequenz precise DNA amplificates. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products. This can be used for easier cloning. However, Pfu is slower and typically requires 1–2 minutes per cycle to amplify 1kb of DNA at 72 °C (compaired to less than 10 seconds using Taq). This is why for most PCR application the cheaper Taq polymerase is still the tool of choice. Conjunction of Pfu and Taq polymerase obtain the fidelity of Pfu with the speed of Taq polymerase activity.

Pfu Polymerases available at genomics-online:

Name Max Target Length (kb) Extension Speed (kb/min)* Fidelity* Proofreading Kits/Mastermix Applications
PFU DNA Polymerase

Y
  • Ideal for high-fidelity amplification
  • Optimal for blunt-end PCR cloning
PFU DNA Polymerase 6 20 20x Y
  • PCR or Primer extension requested High fidelity
  • Gene synthesis
  • Blunt-end PCR Cloning or mutagenesis requested high fidelity
Taq Plus DNA Polymerase 6 1 5x Y ABIN4219187
  • Routine PCR amplification of DNA templates up to 6 kb
  • Suitable for a wide range of PCR assays
  • TA cloning
EasyPfu DNA Polymerase Enzyme 6 (Plasmid: 10) 0,5 18x Y ABIN2953675
  • High fidelity PCR
  • Blunt-end cloning
  • Site-directed mutagenesis
FastPfu DNA Polymerase Enzyme 15 (Plasmid: 20) 2 to 4 54x Y ABIN2953676
  • High fidelity PCR
  • High yield and fast PCR
  • Blunt end cloning
  • Site-directed mutagenesis
  • Complex templates
FastPfu Fly DNA Polymerase Enzyme 15 (Plasmid: 20) up to 6 108x Y ABIN2953676
  • High fidelity PCR
  • High yield and fast PCR
  • Blunt end cloning
  • Site-directed mutagenesis
  • Complex templates
Taq Plus DNA Polymerase 30 Y
  • Ideal for high-fidelity amplification
  • Optimal for blunt-end PCR cloning.

Special polymerases at genomics-online.com

Name Max Target Length (kb) Extension Speed (kb/min)* Fidelity* Proofreading Kits/Mastermix Applications
Bloodirect DNA Polymerase (PCR) 1 1 1x N ABIN4219177
  • hole blood PCR
Bst DNA Polymerase, Large Fragment N
  • Isothermal amplification
  • DNA sequencing of high GC DNA
  • Sequencing from very low amounts of template DNA
  • DNA strand displacement amplification
Bsu DNA Polymerase N
  • Primer labelling
  • Second strand cDNA synthesis
  • DNA strand displacement synthesis
DNA Polymerase I Large (Klenow) Fragment 6x Y
  • DNA blunting by filling-in 5'-overhangs with dNTPs
  • cDNA second-strand synthesis
  • Generate single-stranded DNA probes using random primers
  • Site-directed DNA mutagenesis using synthetic oligonucleotides
  • Dideoxy DNA sequencing of ss &ds DNA
E. coli DNA Polymerase I Y
  • Generation of labeled DNA probes by nick translation
  • Second-strand synthesis of cDNA
Klenow Fragment (3'→5' Exo-) N
  • Dideoxy DNA sequencing DNA templates
  • cDNA second-strand synthesis
  • Generate ssDNA probes using random primers
  • Site-directed DNA mutagenesis using synthetic oligonucleotides
Phi29 DNA Polymerase 70 100x Y
  • Rolling circle amplification(RCA)
  • Multiple displacement amplification(MDA)
  • Whole genome amplification (WGA)
  • DNA template preparation for sequencing
  • Protein-primed DNA amplification
Poly(A) Polymerase, Yeast
  • RNA Manipulation
  • Labelling of RNA with ATP or cordycepin
  • Poly(A) tailing of RNA for cloning or affinity purification
  • Increasing translation of RNA transferred into eukaryotic cells
T4 DNA Polymerase 108x N
  • Generates blunt end DNA by
    filling in 5'-overhangs or/and removing 3'-overhangs
  • High fidelity due to
    strong 3’→5’ exonuclease activity
  • Synthesis of labeled DNA probes
  • Site-specific mutagenesis via primer extension
T7 RNA Polymerase N
  • Synthesis of RNA transcriptsbfor hybridization probes
  • Synthesis of RNA for in vitro translation
  • Synthesis of biologically active mRNA
  • Generate large amounts of labelled or non-labelled RNA

Specificity/Fidelity* Compared to taq polymerase