Highfidelity dna polymerase comparison on difficult genomic dna samples. Phusion highfidelity dna polymerase is comprised of a dnabinding domain fused to a pyrococcuslike proofreading polymerase. Xpert highfidelity dna polymerase grisp research solutions. Phusion highfidelity dna polymerase from new england. Dna polymerases with high fidelity are important for applications in which the dna sequence needs to be correct after amplification. Our quick high fidelity dna polymerase, qhifi dna polymerase tf2000 is a unique blended enzyme mix. It is not crucial for a polymerase with a low error rate it is not crucial for it to have a 3. Highresolution mapping of dna polymerase fidelity using. Highfidelity pcr enzymes are used for applications requiring high accuracy during dna amplification such as cloning, sequencing or mutagenesis. Why are there low molecular weight discrete bands on an agarose gel after a pcr using phusion highfidelity dna polymerase. Manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance. Download a pdf containing pricing for our full product list. The phusion highfidelity dna polymerase should be pipetted carefully and gently as the high glycerol content 50% in the storage buffer may otherwise lead to pipetting errors. Phusion highfidelity dna polymerase, phusion highfidelity pcr kit, phusion highfidelity pcr master mix with hf buffer, phusion highfidelity pcr master mix with gc buffer.
It should be noted that this is the identical substrate used. Phusion highfidelity dna polymerase high performance for. Had dna replication been found to be 100% accurate, darwins theory would have been dead on the spot because there would have been no mechanism to support evolution in asexual. Highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr.
Datasheet for q5 highfidelity dna polymerase m0491. All amplicons obtained using either 25 ng or 25 pg. Modern commercially available highfidelity dna polymerases are derived from either pfu and kod. Detection limit of highfidelity dna polymerases on gdna. What is the error rate of phusion highfidelity dna. The structure of a high fidelity dna polymerase bound to a mismatched nucleotide reveals an ajar intermediate conformation in the nucleotide selection mechanism. Intact genomics ig variety of i7 highfidelity dna polymerase products. Wang department of pathology stanford university school of medicine stanford, california 943055324 enzymatic properties and characteristics that. Dna polymerase fidelity is defined as the ratio of right r to wrong w nucleotide incorporations when drtp and dwtp substrates compete at equal concentrations for primer. Phusion highfidelity dna polymerase for high speed and high fidelity pcr, 52x greater fidelity than taq. Ibi hifi dna polymerase is a high fidelity thermophilic dna polymerase with 5x fewer errors than ibi taq dna polymerase. The thermo scientific phusion highfidelity dna polymerase offers high performance for all major pcr applications.
Phusion highfidelity dna polymerases product listing product overview manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications. Taq plus dna polymerase is a mixture of taq dna polymerase and pfu, a proofreading dna polymerase, which allows for the amplification of long templates, up to 20kb, with high fidelity. Qhifi dna polymerase, quick high fidelity dna polymerase. Proofreading is the primary guardian of dna polymerase fidelity. We have tested i7 highfidelity dna polymerase activity with. Pcr protocol for phusion highfidelity dna polymerase m0530 protocols.
Manufactured and qualitycontrolled at new england biolabs, thermo scientific phusion highfidelity dna polymerase offers both high fidelity and robust performance, and thus can be used for all pcr applications. Dna polymerases are responsible for dna replication, repair and mutation. Lowfidelity dna synthesis by human dna polymerase theta. Thermo scientific phusion highfidelity pcr kit whigh. Highfidelity pcr uses dna polymerases that couple low misincorporation rates with proofreading to give faithful replication of the target dna of interest. Datasheet for phusion highfidelity dna polymerase neb. High fidelity polymerase is based on a blend of taq dna polymerase and a proofreading enzyme specially designed for highly accurate and efficient amplification. Dna replication fidelity friedberg major reference.
It was discovered by thomas kornberg son of arthur kornberg and malcolm gefter in 1970. Mechanistic comparison of highfidelity and errorprone. Dna and other difficult templates for pcr amplification up to 20kb. Thermo scientific phusion highfidelity dna polymerase. Rare base concentration can be tuned to a threshold where a low fidelity dna polymerase lf dnap preferentially creates replication errors but a high fidelity dna polymerase hf dnap still. Phusion dna polymerases thermo fisher scientific us.
Eukaryotes encode three dna polymerases with intrinsic 3. Phusion highfidelity pcr master mix with hf buffer and phusion highfidelity pcr master mix with gc buffer are convenient 2x mixes containing phusion dna polymerase, nucleotides and optimized hf or. Liquid biopsy and detection of tumorassociated mutations in cellfree circulating dna often requires the ability to identify single nucleotide variants at allele frequencies below 0. Fidelity of dna polymerase in dna amplification article pdf available in proceedings of the national academy of sciences 8623. Processivity, fidelity and rate of dna polymerase youtube. The high fidelity of chick embryo dna polymerasegamma polgamma observed during in vitro dna synthesis kunkel, t. What makes highfidelity dna polymerases like phusion or. In this assay, a dna polymerase synthesizes across a 407nt single strand dna gap contained within a 7. High fidelity dna polymerases archives intact genomics. Includes phusion dna polymerase 2 ul 5x phusion hf buffer 5x phusion gc buffer dmso 50 mm mgcl 2 solution both phusion hf buffer and phusion gc buffer provide 1. Processivity, fidelity, and rate of dna replication. Manufactured and qualitycontrolled at new england biolabs, thermo. Pdf fidelity of dna polymerase in dna amplification.
Incorporating an exciting new and patented technology, iproof. How dna polymerases catalyse replication and repair with. A quantitative understanding of high fidelity dna polymerase by tom sharp. Impact of polymerase fidelity on background error rates in. The hifi enzyme contains a 3 5 proofreading exonuclease activity and is designed.
High fidelity pol is based on a blend of taq dna polymerase and a proofreading enzyme specially designed for highly accurate and efficient amplification. An li strain that carries the deep ocean dna polymerase gene from pyrococcus species gbd. Condensed matter a quantitative understanding of high fidelity dna polymerase. Thus, mutations in dna polymerases or changes in their expression could be manifested by alterations in dna replication, in cellcycle. As a result of this unique functional pairing, phusion dna polymerases are capable of generating pcr products with very high accuracy and speed. Dna polymerase iii holoenzyme is the primary enzyme complex involved in prokaryotic dna replication.
Id like to clone a fragment amplified with phusion highfidelity dna polymerase. Pdf dna polymerase fidelity and the polymerase chain. Due to the nature of the phusion highfidelity dna polymerase, the optimal reaction conditions may differ from pcr protocols for standard dna polymerases. Different types of highfidelity dna polymerases are distinguished, including pfu polymerase, a naturally faithful dna polymerase due to its 35 exonuclease activity as well as modified dna polymerases to. Clearly, this parameter alone cannot explain the 100 or more reduced overall fidelity of the y family members compared to the high.
How does a highfidelity polymerase ensure that the correct base is inserted. In one image, we recapitulated the results of the performance comparison and the detection limit of highfidelity dna polymerases on human gdna. The extended strand returns to the polymerase domain, reanneals to the template strand, and replication continues. The following guidelines are provided to ensure successful pcr using phusion dna polymerase. Taq polymerase is the tried and true standard when it comes to pcr, but that doesnt mean its the most accurate polymerase for highsensitivity applications.
Xpert highfidelity dna polymerase is a robust enzyme with enhanced dna binding, resulting in improved processivity, yield, and extremely low errorrate. Incorporating an exciting new and patented technology, iproof dna polymerase brings together a. Dna replication fidelity errol c friedberg, ut southwestern medical center, dallas, texas, usa paula l fischhaber, ut southwestern medical center, dallas, texas, usa dna polymerases are proteins that. Phusion highfidelity dna polymerase thermo fisher scientific. Dna polymerases are required for all dna synthetic processes. Highfidelity dna polymerase comparison using unpurified. Shop online for a wide selection of thermo scientific phusion green highfidelity dna polymerase 2 ul the most accurate thermostable dna polymerase available offering superior. A quantitative understanding of high fidelity dna polymerase. Will phusion highfidelity dna polymerase incorporate dutps. To eliminate spurious mutations in particular for long amplicons, a thermostable dna polymerase with exceptional fidelity is required.