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  1. Article: A Single Amino Acid Change to Taq DNA Polymerase Enables Faster PCR, Reverse Transcription and Strand-Displacement.

    Barnes, Wayne M / Zhang, Zhian / Kermekchiev, Milko B

    Frontiers in bioengineering and biotechnology

    2021  Volume 8, Page(s) 553474

    Abstract: A change of an aspartic acid to asparagine of Taq ( ...

    Abstract A change of an aspartic acid to asparagine of Taq (
    Language English
    Publishing date 2021-01-14
    Publishing country Switzerland
    Document type Journal Article
    ZDB-ID 2719493-0
    ISSN 2296-4185
    ISSN 2296-4185
    DOI 10.3389/fbioe.2020.553474
    Database MEDical Literature Analysis and Retrieval System OnLINE

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  2. Article: Long and accurate PCR.

    Barnes, Wayne M

    CSH protocols

    2006  Volume 2006, Issue 1

    Language English
    Publishing date 2006-06-01
    Publishing country United States
    Document type Journal Article
    DOI 10.1101/pdb.prot4094
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  3. Article: Ribocloning: DNA cloning and gene construction using PCR primers terminated with a ribonucleotide.

    Barnes, Wayne M

    CSH protocols

    2006  Volume 2006, Issue 1

    Language English
    Publishing date 2006-06-01
    Publishing country United States
    Document type Journal Article
    DOI 10.1101/pdb.prot4142
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  4. Article: Streamlined gene assembly PCR.

    Barnes, Wayne M / Frawley, Elaine R

    CSH protocols

    2008  Volume 2008, Page(s) pdb.prot4862

    Abstract: INTRODUCTIONTo construct genes with artificial, designed sequences, the temperature-cycling steps of the PCR process can be used to assemble whole genes and plasmids from identically sized pieces as small as 40 nucleotides. The original protocol for this ...

    Abstract INTRODUCTIONTo construct genes with artificial, designed sequences, the temperature-cycling steps of the PCR process can be used to assemble whole genes and plasmids from identically sized pieces as small as 40 nucleotides. The original protocol for this process entailed two sequential PCR-like reactions. The first cycling protocol of 55 cycles (called "assembly") contained no PCR primers per se; instead, the 40-mers all primed on each other, building up the product gene by extension of 20 bp at each extension step. In the second cycling protocol, real PCR primers were supplied, and 1-2 μL from the first PCR served as the template for an additional 23 cycles (for a total of 78 cycles). Finally, the PCR product was digested by restriction enzymes and gel-purified for cloning. The procedure presented here is a streamlined version of the original methodology, requiring only one round of 20-25 cycles to obtain a single or major product band, as detected by agarose gel analysis. This can be followed directly by cloning without gel-purification of the target DNA.
    Language English
    Publishing date 2008-03-01
    Publishing country United States
    Document type Journal Article
    DOI 10.1101/pdb.prot4862
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  5. Article ; Online: Direct DNA amplification from crude clinical samples using a PCR enhancer cocktail and novel mutants of Taq.

    Zhang, Zhian / Kermekchiev, Milko B / Barnes, Wayne M

    The Journal of molecular diagnostics : JMD

    2010  Volume 12, Issue 2, Page(s) 152–161

    Abstract: PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful ... ...

    Abstract PCR-based clinical and forensic tests often have low sensitivity or even false-negative results caused by potent PCR inhibitors found in blood and soil. It is widely accepted that purification of target DNA before PCR is necessary for successful amplification. In an attempt to overcome PCR inhibition, enhance PCR amplification, and simplify the PCR protocol, we demonstrate improved PCR-enhancing cocktails containing nonionic detergent, l-carnitine, d-(+)-trehalose, and heparin. These cocktails, in combination with two inhibitor-resistant Taq mutants, OmniTaq and Omni Klentaq, enabled efficient amplification of exogenous, endogenous, and high-GC content DNA targets directly from crude samples containing human plasma, serum, and whole blood without DNA purification. In the presence of these enhancer cocktails, the mutant enzymes were able to tolerate at least 25% plasma, serum, or whole blood and as high as 80% GC content templates in PCR reactions. These enhancer cocktails also improved the performance of the novel Taq mutants in real-time PCR amplification using crude samples, both in SYBR Green fluorescence detection and TaqMan assays. The novel enhancer mixes also facilitated DNA amplification from crude samples with various commercial Taq DNA polymerases.
    MeSH term(s) Anticoagulants/metabolism ; DNA/analysis ; DNA/blood ; Heparin/metabolism ; Humans ; Nucleic Acid Amplification Techniques/instrumentation ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction/instrumentation ; Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Taq Polymerase/genetics ; Taq Polymerase/metabolism
    Chemical Substances Anticoagulants ; Heparin (9005-49-6) ; DNA (9007-49-2) ; Taq Polymerase (EC 2.7.7.-)
    Language English
    Publishing date 2010-01-14
    Publishing country United States
    Document type Evaluation Study ; Journal Article ; Research Support, N.I.H., Extramural
    ZDB-ID 2000060-1
    ISSN 1943-7811 ; 1525-1578
    ISSN (online) 1943-7811
    ISSN 1525-1578
    DOI 10.2353/jmoldx.2010.090070
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 5146: Show issues Location:
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  6. Article: Magnesium precipitate hot start method for PCR.

    Barnes, Wayne M / Rowlyk, Katherine R

    Molecular and cellular probes

    2002  Volume 16, Issue 3, Page(s) 167–171

    Abstract: For the amplification of HIV-1 gag gene, and other challenging targets, a simple new hot start PCR protocol is presented which consists simply and entirely of the buffer system. This novel buffer composition and reaction assembly protocol for PCR ... ...

    Abstract For the amplification of HIV-1 gag gene, and other challenging targets, a simple new hot start PCR protocol is presented which consists simply and entirely of the buffer system. This novel buffer composition and reaction assembly protocol for PCR includes magnesium and phosphate combined at high concentration in addition to standard buffer reagents. The resulting magnesium-containing precipitate provides a hot start for PCR, since the magnesium in the precipitate is unavailable to DNA polymerase until thermal cycling. No extra manipulations at the thermal cycler or changes to standard thermal cycling profiles are necessary. Upon normal cycling, the magnesium becomes fully available within the first 3 cycles. The method effectively prevents premature primer extension by several DNA polymerases or mixtures of DNA polymerases tested, including Klentaql, KlentaqLA, Pfu, and full-length wild-type DNA polymerase. Once the precipitate is formed, the hot start buffer is stable and functional for at least a week at -20 degrees, 4 degrees , or 25 degrees. We demonstrate that the method is as effective as a manual hot start (addition of magnesium at or above primers'annealing temperatures) for several target gene amplifications which require a hot start.
    MeSH term(s) Animals ; Buffers ; Cryptosporidium parvum/genetics ; DNA Primers ; DNA-Directed DNA Polymerase ; Gene Products, gag/genetics ; HIV-1/genetics ; Hot Temperature ; Humans ; Magnesium ; Phosphates ; Polymerase Chain Reaction/instrumentation ; Polymerase Chain Reaction/methods ; Tissue Plasminogen Activator/genetics
    Chemical Substances Buffers ; DNA Primers ; Gene Products, gag ; Phosphates ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; Tissue Plasminogen Activator (EC 3.4.21.68) ; Magnesium (I38ZP9992A)
    Language English
    Publishing date 2002-09-12
    Publishing country England
    Document type Journal Article
    ZDB-ID 639082-1
    ISSN 1096-1194 ; 0890-8508
    ISSN (online) 1096-1194
    ISSN 0890-8508
    DOI 10.1006/mcpr.2002.0407
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    Zs.A 2230: Show issues Location:
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  7. Article ; Online: Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples.

    Kermekchiev, Milko B / Kirilova, Lyubka I / Vail, Erika E / Barnes, Wayne M

    Nucleic acids research

    2009  Volume 37, Issue 5, Page(s) e40

    Abstract: Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase ... ...

    Abstract Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10-100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1-1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.
    MeSH term(s) DNA/analysis ; DNA/blood ; Enzyme Inhibitors/blood ; Enzyme Inhibitors/pharmacology ; Fluorescent Dyes ; Humans ; Mutation ; Organic Chemicals ; Polymerase Chain Reaction/methods ; Sequence Deletion ; Soil Microbiology ; Taq Polymerase/antagonists & inhibitors ; Taq Polymerase/genetics ; Taq Polymerase/metabolism ; Time Factors
    Chemical Substances Enzyme Inhibitors ; Fluorescent Dyes ; Organic Chemicals ; SYBR Green I (163795-75-3) ; DNA (9007-49-2) ; Taq Polymerase (EC 2.7.7.-)
    Language English
    Publishing date 2009-02-10
    Publishing country England
    Document type Evaluation Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkn1055
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    Zs.A 1067: Show issues Location:
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  8. Article: Mutants of Taq DNA polymerase resistant to PCR inhibitors allow DNA amplification from whole blood and crude soil samples

    Kermekchiev, Milko B / Kirilova, Lyubka I / Vail, Erika E / Barnes, Wayne M

    Nucleic acids research. 2009 Apr., v. 37, no. 5

    2009  

    Abstract: Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase ... ...

    Abstract Potent PCR inhibitors in blood and soil samples can cause false negative results from PCR-based clinical and forensic tests. We show that the effect of these inhibitors is primarily upon Taq DNA polymerase, since mutational alteration of the polymerase can overcome the inhibition to the extent that no DNA purification is now required. An N-terminal deletion (Klentaq1) is some 10-100-fold inhibition resistant to whole blood compared to full-length, wild-type (w.t.) Taq, which is strongly inhibited by 0.1-1% blood. Further mutations at codon 708, both in Klentaq 1 and Taq, confer enhanced resistance to various inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of intercalating dyes. Blood PCR inhibitors can predominantly reduce the DNA extension speed of the w.t. Taq polymerase as compared to the mutant enzymes. Single-copy human genomic targets are readily amplified from whole blood or crude soil extract, without pretreatment to purify the template DNA, and the allowed increase in dye concentration overcomes fluorescence background and quenching in real-time PCR of blood.
    Keywords DNA ; DNA-directed DNA polymerase ; blood serum ; dyes ; false negative results ; fluorescence ; forensic sciences ; hemoglobin ; humans ; humic acids ; immunoglobulin G ; lactoferrin ; mutants ; mutation ; quantitative polymerase chain reaction ; soil ; soil sampling
    Language English
    Dates of publication 2009-04
    Size p. e40.
    Document type Article
    ZDB-ID 186809-3
    ISSN 0301-5610 ; 0305-1048
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkn1055
    Database NAL-Catalogue (AGRICOLA)

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    Z 79/704: Show issues

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  9. Article ; Online: Cold-sensitive mutants of Taq DNA polymerase provide a hot start for PCR.

    Kermekchiev, Milko B / Tzekov, Anatoly / Barnes, Wayne M

    Nucleic acids research

    2003  Volume 31, Issue 21, Page(s) 6139–6147

    Abstract: Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non- ...

    Abstract Although the thermophilic bacterium Thermus aquaticus grows optimally at 70 degrees C and cannot grow at moderate temperatures, its DNA polymerase I has significant activity at 20-37 degrees C. This activity is a bane to some PCRs, since it catalyzes non-specific priming. We report mutations of Klentaq (an N-terminal deletion variant) DNA polymerase that have markedly reduced activity at 37 degrees C yet retain apparently normal activity at 68 degrees C and resistance at 95 degrees C. The first four of these mutations are clustered on the outside surface of the enzyme, nowhere near the active site, but at the hinge point of a domain that has been proposed to move at each cycle of nucleotide incorporation. We show that the novel cold-sensitive mutants can provide a hot start for PCR and exhibit slightly improved fidelity.
    MeSH term(s) Binding Sites ; Catalytic Domain ; Cold Temperature ; DNA Replication ; Enzyme Stability ; Gene Library ; Hot Temperature ; Mutagenesis/genetics ; Mutation/genetics ; Polymerase Chain Reaction/methods ; Sequence Analysis, DNA ; Taq Polymerase/chemistry ; Taq Polymerase/genetics ; Taq Polymerase/metabolism ; Thermus/enzymology ; Thermus/genetics
    Chemical Substances Taq Polymerase (EC 2.7.7.-)
    Language English
    Publishing date 2003-10-23
    Publishing country England
    Document type Journal Article ; Research Support, U.S. Gov't, P.H.S.
    ZDB-ID 186809-3
    ISSN 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048
    ISSN (online) 1362-4962 ; 1362-4954
    ISSN 0301-5610 ; 0305-1048
    DOI 10.1093/nar/gkg813
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    Zs.A 1067: Show issues Location:
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  10. Article: Mammalian microevolution: Rapid change in mouse mitochondrial DNA.

    Pergams, Oliver R W / Barnes, Wayne M / Nyberg, Dennis

    Nature

    2003  Volume 423, Issue 6938, Page(s) 397

    MeSH term(s) Animals ; Chicago ; DNA, Mitochondrial/genetics ; Evolution, Molecular ; Haplotypes/genetics ; Mice ; Peromyscus/genetics ; Skin ; Time Factors
    Chemical Substances DNA, Mitochondrial
    Language English
    Publishing date 2003-05-22
    Publishing country England
    Document type Journal Article
    ZDB-ID 120714-3
    ISSN 1476-4687 ; 0028-0836
    ISSN (online) 1476-4687
    ISSN 0028-0836
    DOI 10.1038/423397a
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