The Experts below are selected from a list of 113424 Experts worldwide ranked by ideXlab platform
Michael J Hurley - One of the best experts on this subject based on the ideXlab platform.
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enhanced allele specific pcr discrimination in snp genotyping using 3 locked nucleic acid lna Primers
Human Mutation, 2003Co-Authors: David Latorra, Krista Campbell, Andreas Wolter, Michael J HurleyAbstract:The specificity and reliability of locked nucleic acid (LNA) substitution at the 3′ position of allele-specific PCR (AS-PCR) Primers for SNP detection was investigated in direct comparison to DNA Primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3′ LNA mismatch combinations were tested in triplicate with the plasmid target. LNA Primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA Primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3′ LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3′ LNA Primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3′ LNA Primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications. Hum Mutat 22:79–85, 2003.© 2003 Wiley-Liss, Inc.
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enhanced allele specific pcr discrimination in snp genotyping using 3 locked nucleic acid lna Primers
Human Mutation, 2003Co-Authors: David Latorra, Krista Campbell, Andreas Wolter, Michael J HurleyAbstract:The specificity and reliability of locked nucleic acid (LNA) substitution at the 3' position of allele-specific PCR (AS-PCR) Primers for SNP detection was investigated in direct comparison to DNA Primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3' LNA mismatch combinations were tested in triplicate with the plasmid target. LNA Primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA Primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3' LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3' LNA Primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3' LNA Primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.
Bernard Moss - One of the best experts on this subject based on the ideXlab platform.
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products and substrate template usage of vaccinia virus DNA primase
Virology, 2009Co-Authors: Frank S. De Silva, Nir Paran, Bernard MossAbstract:Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded varphiX174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which (32)P was transferred from [alpha-(32)P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA Primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.
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Products and substrate/template usage of vaccinia virus DNA primase
Virology, 2008Co-Authors: Frank S. De Silva, Nir Paran, Bernard MossAbstract:Vaccinia virus encodes a 90-kDa protein conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded varphiX174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which (32)P was transferred from [alpha-(32)P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA Primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.
David Latorra - One of the best experts on this subject based on the ideXlab platform.
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enhanced allele specific pcr discrimination in snp genotyping using 3 locked nucleic acid lna Primers
Human Mutation, 2003Co-Authors: David Latorra, Krista Campbell, Andreas Wolter, Michael J HurleyAbstract:The specificity and reliability of locked nucleic acid (LNA) substitution at the 3′ position of allele-specific PCR (AS-PCR) Primers for SNP detection was investigated in direct comparison to DNA Primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3′ LNA mismatch combinations were tested in triplicate with the plasmid target. LNA Primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA Primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3′ LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3′ LNA Primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3′ LNA Primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications. Hum Mutat 22:79–85, 2003.© 2003 Wiley-Liss, Inc.
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enhanced allele specific pcr discrimination in snp genotyping using 3 locked nucleic acid lna Primers
Human Mutation, 2003Co-Authors: David Latorra, Krista Campbell, Andreas Wolter, Michael J HurleyAbstract:The specificity and reliability of locked nucleic acid (LNA) substitution at the 3' position of allele-specific PCR (AS-PCR) Primers for SNP detection was investigated in direct comparison to DNA Primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3' LNA mismatch combinations were tested in triplicate with the plasmid target. LNA Primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA Primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3' LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3' LNA Primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3' LNA Primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.
Tahir H. Tahirov - One of the best experts on this subject based on the ideXlab platform.
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activity and fidelity of human DNA polymerase α depend on primer structure
Journal of Biological Chemistry, 2018Co-Authors: Andrey G. Baranovskiy, Nigar D. Babayeva, Yinbo Zhang, Youri I. Pavlov, Vincent N Duong, Karen S Anderson, Tahir H. TahirovAbstract:DNA polymerase α (Polα) plays an important role in genome replication. In a complex with primase, Polα synthesizes chimeric RNA-DNA Primers necessary for replication of both chromosomal DNA strands. During RNA primer extension with deoxyribonucleotides, Polα needs to use double-stranded helical substrates having different structures. Here, we provide a detailed structure-function analysis of human Polα's interaction with dNTPs and DNA templates primed with RNA, chimeric RNA-DNA, or DNA. We report the crystal structures of two ternary complexes of the Polα catalytic domain containing dCTP, a DNA template, and either a DNA or an RNA primer. Unexpectedly, in the ternary complex with a DNA:DNA duplex and dCTP, the "fingers" subdomain of Polα is in the open conformation. Polα induces conformational changes in the DNA and hybrid duplexes to produce the universal double helix form. Pre-steady-state kinetic studies indicated for both duplex types that chemical catalysis rather than product release is the rate-limiting step. Moreover, human Polα extended DNA Primers with higher efficiency but lower processivity than it did with RNA and chimeric Primers. Polα has a substantial propensity to make errors during DNA synthesis, and we observed that its fidelity depends on the type of sugar at the primer 3'-end. A detailed structural comparison of Polα with other replicative DNA polymerases disclosed common features and some differences, which may reflect the specialization of each polymerase in genome replication.
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Mechanism of Concerted RNA-DNA Primer Synthesis by the Human Primosome.
The Journal of biological chemistry, 2016Co-Authors: Andrey G. Baranovskiy, Nigar D. Babayeva, Yinbo Zhang, Yoshiaki Suwa, Youri I. Pavlov, Tahir H. TahirovAbstract:The human primosome, a 340-kilodalton complex of primase and DNA polymerase α (Polα), synthesizes chimeric RNA-DNA Primers to be extended by replicative DNA polymerases δ and ϵ. The intricate mechanism of concerted primer synthesis by two catalytic centers was an enigma for over three decades. Here we report the crystal structures of two key complexes, the human primosome and the C-terminal domain of the primase large subunit (p58C) with bound DNA/RNA duplex. These structures, along with analysis of primase/polymerase activities, provide a plausible mechanism for all transactions of the primosome including initiation, elongation, accurate counting of RNA primer length, primer transfer to Polα, and concerted autoregulation of alternate activation/inhibition of the catalytic centers. Our findings reveal a central role of p58C in the coordinated actions of two catalytic domains in the primosome and ultimately could impact the design of anticancer drugs.
Krista Campbell - One of the best experts on this subject based on the ideXlab platform.
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enhanced allele specific pcr discrimination in snp genotyping using 3 locked nucleic acid lna Primers
Human Mutation, 2003Co-Authors: David Latorra, Krista Campbell, Andreas Wolter, Michael J HurleyAbstract:The specificity and reliability of locked nucleic acid (LNA) substitution at the 3′ position of allele-specific PCR (AS-PCR) Primers for SNP detection was investigated in direct comparison to DNA Primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3′ LNA mismatch combinations were tested in triplicate with the plasmid target. LNA Primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA Primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3′ LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3′ LNA Primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3′ LNA Primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications. Hum Mutat 22:79–85, 2003.© 2003 Wiley-Liss, Inc.
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enhanced allele specific pcr discrimination in snp genotyping using 3 locked nucleic acid lna Primers
Human Mutation, 2003Co-Authors: David Latorra, Krista Campbell, Andreas Wolter, Michael J HurleyAbstract:The specificity and reliability of locked nucleic acid (LNA) substitution at the 3' position of allele-specific PCR (AS-PCR) Primers for SNP detection was investigated in direct comparison to DNA Primers. Both plasmid and human genomic DNA templates were examined in this study. All possible DNA and 3' LNA mismatch combinations were tested in triplicate with the plasmid target. LNA Primers yield consistently low amounts of mismatch products with all base combinations, whereas certain mismatches with DNA Primers generate strong false positive amplicons. Amplified human SNP alleles within the cystic fibrosis (CFTR) gene were analyzed in AS-PCR by gel analysis and real-time fluorescence generation. A 3' LNA residue in the primer at the SNP site improves allelic discrimination and functions under a wide window of PCR conditions. We demonstrate increased AS-PCR specificity with comparable sensitivity using 3' LNA Primers in gel electrophoresis and real-time detection experiments. This increase in AS-PCR discrimination with 3' LNA Primers should facilitate the use of this simple, rapid, and inexpensive technique for SNP genotyping applications.