The Experts below are selected from a list of 1686 Experts worldwide ranked by ideXlab platform
Natasha Paul - One of the best experts on this subject based on the ideXlab platform.
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Hot Start PCR.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Natasha Paul, Jonathan ShumAbstract:Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components such as magnesium ion, DNA polymerase, oligonucleotide primers, and dNTPs. Herein, five different Hot Start activation protocols are presented. The first method presents the use of barriers as a means of segregating key reaction components until a Hot Start activation step. The second and third protocols demonstrate Hot Start approaches to block DNA polymerase activity through the use of anti-DNA polymerase antibodies and accessory proteins, respectively. The fourth and fifth protocols utilize thermolabile chemical modifications to the oligonucleotide primers and dNTPs. The results presented demonstrate that all protocols significantly improve the specificity of traditional thermal cycling protocols.
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Heat-activatable primers for Hot-Start PCR and Hot-Start one-step RT-PCR: endpoint and real-time experiments.
Current protocols in molecular biology, 2009Co-Authors: Elena Hidalgo Ashrafi, Natasha PaulAbstract:Hot-Start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes Hot-Start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phospHotriester groups onto the 3' end of the primer allows for primer-based Hot-Start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a Hot-Start polymerase suitable for your application is not available.
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Current Protocols in Molecular Biology - Heat-activatable primers for Hot-Start PCR and Hot-Start one-step RT-PCR: endpoint and real-time experiments.
Current Protocols in Molecular Biology, 2009Co-Authors: Elena Hidalgo Ashrafi, Natasha PaulAbstract:Hot-Start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes Hot-Start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phospHotriester groups onto the 3' end of the primer allows for primer-based Hot-Start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a Hot-Start polymerase suitable for your application is not available.
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Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance
Nucleic acids research, 2008Co-Authors: Alexandre Lebedev, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Victor A. Timoshchuk, Kei Miyagi, Jack Kellum, Gerald ZonAbstract:The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phospHotriester (PTE) modification groups at 3’-terminal and 3’-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in onestep reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.
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Heat Activatable 3′-modified dNTPs: Synthesis and Application for Hot Start PCR
Nucleic Acids Symposium Series, 2008Co-Authors: Inna Koukhareva, Huang Haoqiang, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Alexandre LebedevAbstract:Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.
Elena Hidalgo Ashrafi - One of the best experts on this subject based on the ideXlab platform.
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Heat-activatable primers for Hot-Start PCR and Hot-Start one-step RT-PCR: endpoint and real-time experiments.
Current protocols in molecular biology, 2009Co-Authors: Elena Hidalgo Ashrafi, Natasha PaulAbstract:Hot-Start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes Hot-Start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phospHotriester groups onto the 3' end of the primer allows for primer-based Hot-Start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a Hot-Start polymerase suitable for your application is not available.
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Current Protocols in Molecular Biology - Heat-activatable primers for Hot-Start PCR and Hot-Start one-step RT-PCR: endpoint and real-time experiments.
Current Protocols in Molecular Biology, 2009Co-Authors: Elena Hidalgo Ashrafi, Natasha PaulAbstract:Hot-Start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes Hot-Start PCR protocols which utilize primers containing temperature-sensitive modifications. The introduction of 4-oxo-tetradecyl (OXT) phospHotriester groups onto the 3' end of the primer allows for primer-based Hot-Start PCR that is amenable for use in a number of PCR-based applications. The protocols described in this unit utilize OXT-modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one-step reverse-transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a Hot-Start polymerase suitable for your application is not available.
Jonathan Shum - One of the best experts on this subject based on the ideXlab platform.
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Hot Start PCR.
Methods in molecular biology (Clifton N.J.), 2010Co-Authors: Natasha Paul, Jonathan ShumAbstract:Hot Start activation approaches are increasingly being used to improve the performance of PCR. Since the inception of Hot Start as a means of blocking DNA polymerase extension at lower temperatures, a number of approaches have been developed that target the essential reaction components such as magnesium ion, DNA polymerase, oligonucleotide primers, and dNTPs. Herein, five different Hot Start activation protocols are presented. The first method presents the use of barriers as a means of segregating key reaction components until a Hot Start activation step. The second and third protocols demonstrate Hot Start approaches to block DNA polymerase activity through the use of anti-DNA polymerase antibodies and accessory proteins, respectively. The fourth and fifth protocols utilize thermolabile chemical modifications to the oligonucleotide primers and dNTPs. The results presented demonstrate that all protocols significantly improve the specificity of traditional thermal cycling protocols.
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Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance
Nucleic acids research, 2008Co-Authors: Alexandre Lebedev, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Victor A. Timoshchuk, Kei Miyagi, Jack Kellum, Gerald ZonAbstract:The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phospHotriester (PTE) modification groups at 3’-terminal and 3’-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in onestep reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.
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Heat Activatable 3′-modified dNTPs: Synthesis and Application for Hot Start PCR
Nucleic Acids Symposium Series, 2008Co-Authors: Inna Koukhareva, Huang Haoqiang, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Alexandre LebedevAbstract:Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.
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Heat activatable 3'-modified dNTPs: synthesis and application for Hot Start PCR.
Nucleic acids symposium series (2004), 2008Co-Authors: Inna Koukhareva, Huang Haoqiang, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Alexandre V LebedevAbstract:Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.
Alexandre Lebedev - One of the best experts on this subject based on the ideXlab platform.
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Heat-activatable primers for Hot-Start PCR: oligonucleotide synthesis and basic PCR setup.
Current protocols in nucleic acid chemistry, 2009Co-Authors: Alexandre LebedevAbstract:2'-Deoxyribonucleoside-3'-O-(4-oxotetradec-1-yl) phosphoramidites (OXT phosphoramidites) are used to prepare modified oligodeoxyribonucleotide primers containing heat cleavable OXT phospHotriester protecting groups at 3'-ultimate and penultimate internucleotide linkages. The OXT-modified primers significantly improve performance of the polymerase chain reaction (PCR) compared to standard DNA primers by substantially reducing or eliminating the accumulation of PCR artifacts such as dimerized primers and misprimed amplicons. Basic protocols for synthesis of OXT-modified oligonucleotide primers and for performing Hot-Start PCR are described.
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Current Protocols in Nucleic Acid Chemistry - Heat-activatable primers for Hot-Start PCR: oligonucleotide synthesis and basic PCR setup.
Current Protocols in Nucleic Acid Chemistry, 2009Co-Authors: Alexandre LebedevAbstract:2′-Deoxyribonucleoside-3′-O-(4-oxotetradec-1-yl) phosphoramidites (OXT phosphoramidites) are used to prepare modified oligodeoxyribonucleotide primers containing heat cleavable OXT phospHotriester protecting groups at 3′-ultimate and penultimate internucleotide linkages. The OXT-modified primers significantly improve performance of the polymerase chain reaction (PCR) compared to standard DNA primers by substantially reducing or eliminating the accumulation of PCR artifacts such as dimerized primers and misprimed amplicons. Basic protocols for synthesis of OXT-modified oligonucleotide primers and for performing Hot-Start PCR are described. Curr. Protoc. Nucleic Acid Chem. 38:4.35.1-4.35.17. © 2009 by John Wiley & Sons, Inc. Keywords: PCR; Hot-Start PCR; primer dimer; mispriming; 3′-phosphoramidite; phospHotriester; oligonucleotide; DNA polymerase
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Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance
Nucleic acids research, 2008Co-Authors: Alexandre Lebedev, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Victor A. Timoshchuk, Kei Miyagi, Jack Kellum, Gerald ZonAbstract:The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phospHotriester (PTE) modification groups at 3’-terminal and 3’-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in onestep reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.
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Heat Activatable 3′-modified dNTPs: Synthesis and Application for Hot Start PCR
Nucleic Acids Symposium Series, 2008Co-Authors: Inna Koukhareva, Huang Haoqiang, Joyclyn Yee, Jonathan Shum, Natasha Paul, Richard I Hogrefe, Alexandre LebedevAbstract:Several 3'-ether and 3'-ester derivatives of 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were prepared. These dNTP derivatives were not substrates for DNA polymerase and did not support primer extension at room temperature. However, by short pre-heating to 95 degrees C in PCR buffer, these 3'-modified dNTPs can be converted to corresponding unmodified natural dNTPs that efficiently support PCR amplification. The analysis of PCR products obtained with 3'-modified dNTPs revealed a significant improvement in PCR performance resulting in higher amplicon yield and reduced formation of off-target products (mis-priming and primer dimer). Among the studied 3'-modified dNTPs, the 3'-tetrahydrofuranyl derivatives showed the best results.
David E. Birch - One of the best experts on this subject based on the ideXlab platform.
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Simplified Hot Start PCR
Nature, 1996Co-Authors: David E. Birch, Lori A. Kolmodin, J. Wong, G. A. Zangenberg, Michael Anthony Zoccoli, N. Mckinney, K. K. Y. YoungAbstract:The use of a thermally activated DNA polymerase PCR gives improved specificity, sensitivity and product yield without additives or extra process steps.
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Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.
Nucleic acids research, 1992Co-Authors: Quin Chou, David E. Birch, Marion Russell, Jonathan Raymond, Will BlochAbstract:A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).