A thermostable DNA polymerase which possesses an associated 3'-to-5' exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10% of the number of mutations obtained from similar amplifications performed with Taq DNA polymerase. The PCR fidelity assay is based on the amplification and cloning of lacI, lacO and lacZ alpha gene sequences (lacIOZ alpha) using either Pfu or Taq DNA polymerase. Certain mutations within the lacI gene inactivate the Lac repressor protein and permit the expression of beta Gal. When plated on a chromogenic substrate, these LacI- mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the 182 known detectable sites of the lacI gene was 1.6 x 10(-6) for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x 10(-5) error rate for Taq DNA polymerase, after approx. 10(5)-fold amplification.
"Improving fidelity with the goal of producing error-free PCR products has been a substantial challenge and has many bioscience companies still looking for a solution. This has, however, led to the discovery of a number of new thermostable enzymes that have error rates improved from the original Taq polymerase   . Yet with all these improvements the commercially marketed polymerases with the highest fidelity still misincorporate 1 base out of 16,000 up from 1 base out of 1600 seen with Taq polymerase . "
[Show abstract][Hide abstract] ABSTRACT: DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the í µí»¼ polymerase, the í µí»½ sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of í µí»¼, í µí»½ and DnaX (í µí¼ and í µí»¾), í µí»¿ and í µí»¿ í® í° components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the í µí»¼ polymerase was found to be unstable at temperatures above 65 ∘ C. We were able to increase the thermostability of the pol III HE to 98 ∘ C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.
"The fidelity of DNA synthesis in vitro is markedly affected by the reaction condition. However, the archaeal family B enzymes generally perform more accurate DNA synthesis as compared with Taq polymerase (Cariello et al., 1991; Ling et al., 1991; Lundberg et al., 1991; Mattila et al., 1991), suggesting that the strong 3′–5′ exonuclease activities of the hyperthermophilic family B polymerase in vitro affect the fidelity of PCR. "
[Show abstract][Hide abstract] ABSTRACT: DNA polymerase is a ubiquitous enzyme that synthesizes complementary DNA strands according to the template DNA in living cells. Multiple enzymes have been identified from each organism, and the shared functions of these enzymes have been investigated. In addition to their fundamental role in maintaining genome integrity during replication and repair, DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, labeling, mutagenesis, and other purposes. The fundamental ability of DNA polymerases to synthesize a deoxyribonucleotide chain is conserved. However, the more specific properties, including processivity, fidelity (synthesis accuracy), and substrate nucleotide selectivity, differ among the enzymes. The distinctive properties of each DNA polymerase may lead to the potential development of unique reagents, and therefore searching for novel DNA polymerase has been one of the major focuses in this research field. In addition, protein engineering techniques to create mutant or artificial DNA polymerases have been successfully developing powerful DNA polymerases, suitable for specific purposes among the many kinds of DNA manipulations. Thermostable DNA polymerases are especially important for PCR-related techniques in molecular biology. In this review, we summarize the history of the research on developing thermostable DNA polymerases as reagents for genetic manipulation and discuss the future of this research field.
Frontiers in Microbiology 08/2014; 5:465. DOI:10.3389/fmicb.2014.00465 · 3.99 Impact Factor
"A widely used method for whole genome amplification is multiple displacement amplification (MDA); MDA relies on priming of target DNA with random primers and the use of the strand-displacing φ29 polymerase to amplify all of the DNA in a given sample
[1–3]. φ29 DNA polymerase is a highly processive, strand-displacing polymerase with a very low error rate of 1 in 106-107 nucleotides
[4, 5]; the error rates of Taq polymerase and Pfu polymerase, both commonly used in PCR are 3 in 104 and 3 in 106, respectively
[6, 7]. Recently a method for the whole genome amplification of DNA from single cells called MALBAC was reported to perform better than MDA
[Show abstract][Hide abstract] ABSTRACT: Background
Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA).
A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome.
The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.
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