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Polymerases

Julian Pampel

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:

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

The structure of DNA polymerase is highly conserved, meaning their catalytic subunits vary very little from one species to another, irrespective of how their domains are structured. This highly conserved structure usually indicates that the cellular functions they perform are crucial and irreplaceable and therefore require rigid maintenance to ensure their evolutionary advantage.

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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.com:

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
EasyTaq® DNA Polymerase for PAGE (with 2.5 mM dNTPs) / (without 2.5 mM dNTPs)	3	1 to 2		Y	ABIN5519407	Short fragment PCR
TransStart® Taq DNA Polymerase	4	1 to 2	1x	N	ABIN5519400	Routine PCR Colony PCR
TransFast® Taq DNA Polymerase	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
TransTaq®-T DNA Polymerase	8	1 to 2	18x	Y	ABIN5519418	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
TransTaq® DNA Polymerase High Fidelity(HiFi) (with 2.5 mM dNTPs) / (without 2.5 mM dNTPs)	15	1 to 2	18x	Y	genomic DNA λDNA, cDNA and plasmid DNA	Complex templates GC/AT rich templates Long PCR High yield PCR
TransStart® TopTaq DNA Polymerase	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
TransStart® KD Plus DNA Polymerase (with 2.5 mM dNTPs) / (without 2.5 mM dNTPs)	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 (with 2.5 mM dNTPs) / (without 2.5 mM dNTPs)	6 (Plasmid: 10)	0,5	18x	Y	ABIN5519398	High fidelity PCR Blunt-end cloning Site-directed mutagenesis
TransStart® FastPfu DNA Polymerase (with 2.5 mM dNTPs) / (without 2.5 mM dNTPs)	15 (Plasmid: 20)	2 to 4	54x	Y	ABIN5519412	High fidelity PCR High yield and fast PCR Blunt end cloning Site-directed mutagenesis Complex templates
TransStart® FastPfu Fly DNA Polymerase (with 2.5 mM dNTPs) / (without 2.5 mM dNTPs)	15 (Plasmid: 20)	up to 6	108x	Y	ABIN5519410	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.

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Special polymerases at genomics-online.com

In this list you can find polymerase designed for special experimental demands e.g. different DNA & RNA manipulations, a PCR made with blood as direct template or very long ssDNA templates. Please browse our product portfolio. If you need help with finding the right product, please contact our competent customer support via live chat, email or phone.

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 dsDNA
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

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