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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 ; 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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  3. 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
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1067: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
    bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular
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  4. 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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    In stock of ZB MED Bonn / Germany

    Z 79/704: Show issues

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  5. 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
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 1067: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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  6. Article ; Online: A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.

    Wu, Eugene Y / Walsh, Amanda R / Materne, Emma C / Hiltner, Emily P / Zielinski, Bryan / Miller, Bill R / Mawby, Lily / Modeste, Erica / Parish, Carol A / Barnes, Wayne M / Kermekchiev, Milko B

    Biochemistry

    2015  Volume 54, Issue 3, Page(s) 881–889

    Abstract: Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity ...

    Abstract Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5'-to-3' exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E-DNA-ddNTP) and binary (E-DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5'-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.
    MeSH term(s) Cold Temperature ; Crystallography, X-Ray ; DNA/chemistry ; Isoleucine/genetics ; Kinetics ; Leucine/genetics ; Models, Molecular ; Molecular Dynamics Simulation ; Mutant Proteins/chemistry ; Mutation/genetics ; Nucleotides/chemistry ; Taq Polymerase/chemistry ; Taq Polymerase/genetics
    Chemical Substances Mutant Proteins ; Nucleotides ; Isoleucine (04Y7590D77) ; DNA (9007-49-2) ; Taq Polymerase (EC 2.7.7.-) ; Leucine (GMW67QNF9C)
    Language English
    Publishing date 2015-01-09
    Publishing country United States
    Document type Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021/bi501198f
    Database MEDical Literature Analysis and Retrieval System OnLINE

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 217: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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  7. Article: A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase

    Wu, Eugene Y / Walsh Amanda R / Materne Emma C / Hiltner Emily P / Zielinski Bryan / Miller Bill R / Mawby Lily / Modeste Erica / Parish Carol A / Barnes Wayne M / Kermekchiev Milko B

    Biochemistry. 2015 Jan. 27, v. 54, no. 3

    2015  

    Abstract: Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity ...

    Abstract Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.
    Keywords DNA ; DNA-directed DNA polymerase ; Thermus aquaticus ; X-ray diffraction ; active sites ; ambient temperature ; cold ; isoleucine ; leucine ; mutation ; phenotype ; phenylalanine ; pyrimidines ; soaking
    Language English
    Dates of publication 2015-0127
    Size p. 881-889.
    Publishing place American Chemical Society
    Document type Article
    ZDB-ID 1108-3
    ISSN 1520-4995 ; 0006-2960
    ISSN (online) 1520-4995
    ISSN 0006-2960
    DOI 10.1021%2Fbi501198f
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Cologne/Königswinter

    Zs.A 217: Show issues Location:
    Je nach Verfügbarkeit (siehe Angabe bei Bestand)
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  8. Article: A Conservative Isoleucine to Leucine Mutation Causes Major Rearrangements and Cold Sensitivity in KlenTaq1 DNA Polymerase

    Wu, Eugene Y. / Walsh Amanda R.author / Materne Emma C.author / Hiltner Emily P.author / Zielinski Bryanauthor / Miller Bill R.author / Mawby Lilyauthor / Modeste Ericaauthor / Parish Carol A.author / Barnes Wayne M.authorDepartment of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, Missouri 63110, United States / Kermekchiev Milko B.authorDNA Polymerase Technology, Inc., St. Louis, Missouri 63104, United States
    Language English
    Document type Article
    Database AGRIS - International Information System for the Agricultural Sciences and Technology

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