Presentation is loading. Please wait.

Presentation is loading. Please wait.

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction  Karen S. Gustafson  The Journal.

Published byAllison Watson Modified over 5 years ago

Similar presentations

Presentation on theme: "Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction  Karen S. Gustafson  The Journal."— Presentation transcript:

1 Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction  Karen S. Gustafson  The Journal of Molecular Diagnostics  Volume 10, Issue 1, Pages (January 2008) DOI: /jmoldx Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Schematic representation of methylated CpG sites in the IGSF4 promoter. The positions of the DNA and LNA primers and probes relative to the transcription start site are shown. The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 LNA primers enhance analytical specificity of QMSP with SYBR Green I. A–C: QMSP performed with IGSF4 FM4 and IGSF4 RM4 DNA primers and MC DNA template (black triangles), UC DNA template (black squares), NTC (black circles). Amplification plots (A) of reactions with DNA primers performed at an annealing temperature (Ta) of 60°C and corresponding melt curves (B) are shown. The increased fluorescence detected in reactions with IGSF4 FM4 and IGSF4 RM4 DNA primers and MC DNA template (black triangles) represents a single amplified product with a Tm of 79.2°C (melt curve). Nonspecific products with lower Tms were generated with DNA primers in reactions with UC DNA (black squares) and NTC (black circles). C: Increasing the Ta to 65°C resulted in delayed Ct values of >5 for the nonspecific products; however, they were not completely eliminated. D–F: QMSP performed with IGSF4 FM4L and IGSF4 RM4L LNA primers and MC DNA template (black triangles), UC DNA template (black squares), NTC (black circles). Amplification plots (D) of reactions with LNA primers performed at Ta of 60°C and corresponding melt curves (E) are shown. Use of LNA primers resulted in delayed Ct values of ∼7 for the nonspecific products (compare A to D). Increasing the Ta from 60 to 65°C completely eliminated the formation of nonspecific products in UC DNA and NTC samples (compare D to F). The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 QMSP with LNA primers eliminates mispriming from unmethylated DNA templates. A and B: QMSP performed with IGSF4 EXF and IGSF4 EXR DNA primers and M-IGSF4-pGEM plasmid (white triangles) or U-IGSF4-pGEM plasmid (white squares) DNA templates. Amplification plots (A) and corresponding melt curves (B) are shown. A single product was generated from each plasmid template with a different Tm depending on GC content. Based on Ct values, equivalent amounts of M-IGSF4-pGEM and U-IGSF4-pGEM plasmid templates were added to the reactions with IGSF4 DNA and LNA primers. C and D: QMSP performed with IGSF4 FM4 and IGSF4 RM4 DNA primers and equivalent amounts of M-IGSF4-pGEM plasmid (white triangles) or U-IGSF4-pGEM plasmid (white squares), 50 ng of MC DNA (black triangles) or UC DNA (black squares) templates, or NTC (black circles). Amplification plots (C) and corresponding melt curves (D) are shown. Nonspecific products were formed with both the U-IGSF4-pGEM and UC DNA template that had identical Tms; Ct values were delayed compared to methylated templates. E and F: QMSP performed with IGSF4 FM4L and RM4L LNA primers and equivalent amounts of M-IGSF4-pGEM plasmid (white triangles) or U-IGSF4-pGEM plasmid (white squares), 50 ng of MC DNA (black triangles) or UC DNA (black squares) templates, or NTC (black circles). Amplification plots (E) and corresponding melt curves (F) are shown. Nonspecific products generated with unmethylated templates and NTC were eliminated. Representative experiment; data shown are the average of duplicate samples. All reactions were performed at a Ta of 63°C. The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5 Figure 4 Agarose gel electrophoresis of QMSP products. Aliquots (10 μl) of samples from QMSP performed with IGSF4 FM4 and IGSF4 RM4 DNA primers (lanes 1 to 5) and IGSF4 FM4L and IGSF4 RM4L LNA primers (lanes 6 to 10) were analyzed by gel electrophoresis. DNA templates used in the reactions were as follows: NTC (lanes 1 and 6), U-IGSF4-pGEM (lanes 2 and 7), UC DNA (lanes 3 and 8), M-IGSF4-pGEM (lanes 4 and 9), and MC DNA (lanes 5 and 10). The nonspecific products formed using DNA primers and unmethylated DNA templates are of the same size as the specific products formed with the methylated templates (99 bp; indicated by the double asterisk), whereas the nonspecific product (primer dimer) formed in the NTC reaction is smaller in size (indicated by the asterisk). The enhanced specificity of LNA primers eliminated the formation of nonspecific products in reactions with unmethylated DNA templates and NTC (lanes 6 to 8). M, 50-bp molecular weight marker. The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

6 Figure 5 Analytical performance of QMSP with SYBR Green I and LNA primers. Standard curve plot showing Ct versus initial quantity of fivefold serially diluted (50 ng to 16 pg) MC DNA with excess UC DNA, in which the total amount of DNA was kept constant at 50 ng, demonstrates that the analytical sensitivity of the assay is 16 pg. The slope of −3.393 reflects efficient PCR amplification, and the correlation coefficient (R2) of indicates that the assay was linear throughout the 3125-fold range of template concentrations. Representative experiment; data shown are the average of duplicate samples. The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

7 Figure 6 Comparison of the amplification efficiency between QMSP performed with DNA and LNA fluorogenic probes. QMSP was performed at a Ta of 65°C with 50 ng of MC DNA template using IGSF4 FM4L and IGSF4 RM5L LNA primers and either the FM DNA probe (black triangles) or FML LNA probe (black squares). Reactions with the LNA probe show more efficient amplification of methylated alleles with a lower Ct value (Ct = 25.62) and higher fluorescence signal (dRn last value = 4.53) compared to reactions with the DNA probe (Ct = 26.63, dRn last = 2.29). Representative experiment; data shown are the average of duplicate samples. The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

8 Figure 7 Analytical performance of QMSP with LNA fluorogenic probe and primers. Standard curve plot showing Ct versus initial quantity of fivefold serially diluted (50 ng to 16 pg) MC DNA with excess UC DNA, in which the total amount of DNA was kept constant at 50 ng, demonstrates that the analytical sensitivity of the assay is 16 pg. The slope of −3.279 reflects efficient PCR amplification and the correlation coefficient (R2) of shows that the assay is linear throughout the 3125-fold range of template concentrations. Representative experiment; data shown are the average of duplicate samples. The Journal of Molecular Diagnostics  , 33-42DOI: ( /jmoldx ) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Download ppt "Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction  Karen S. Gustafson  The Journal."

Similar presentations

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis  Philippe Szankasi, David.

Clinical Laboratory Analysis of Immunoglobulin Heavy Chain Variable Region Genes for Chronic Lymphocytic Leukemia Prognosis  Philippe Szankasi, David.
A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations  Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.

A Melting Curve Analysis–Based PCR Assay for One-Step Genotyping of β- Thalassemia Mutations  Fu Xiong, Qiuying Huang, Xiaoyun Chen, Yuqiu Zhou, Xinhua.
Principles of Real-Time Quantitative PCR Techniques

Principles of Real-Time Quantitative PCR Techniques
Detection of Exon 12 Mutations in the JAK2 Gene

Detection of Exon 12 Mutations in the JAK2 Gene
Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq  Zhian Zhang, Milko B. Kermekchiev, Wayne.

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq  Zhian Zhang, Milko B. Kermekchiev, Wayne.
Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction  James.

Pre-Clinical Validation of a Novel, Highly Sensitive Assay to Detect PML-RARα mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction  James.
Todd S. Laughlin, Michael W. Becker, Jane L. Liesveld, Deborah A

Todd S. Laughlin, Michael W. Becker, Jane L. Liesveld, Deborah A
One-Step Ligation on RNA Amplification for the Detection of Point Mutations  Lei Zhang, Jingjing Wang, Mia Coetzer, Stephanie Angione, Rami Kantor, Anubhav.

One-Step Ligation on RNA Amplification for the Detection of Point Mutations  Lei Zhang, Jingjing Wang, Mia Coetzer, Stephanie Angione, Rami Kantor, Anubhav.
Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq  Zhian Zhang, Milko B. Kermekchiev, Wayne.

Direct DNA Amplification from Crude Clinical Samples Using a PCR Enhancer Cocktail and Novel Mutants of Taq  Zhian Zhang, Milko B. Kermekchiev, Wayne.
Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons 

Rapid Detection of the Epidermal Growth Factor Receptor Mutation in Non-Small-Cell Lung Cancer for Analysis of Acquired Resistance Using Molecular Beacons 
Gene quantification using real-time quantitative PCR

Gene quantification using real-time quantitative PCR
Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction  Kamesh.

Multiplex Detection of Ehrlichia and Anaplasma Species Pathogens in Peripheral Blood by Real-Time Reverse Transcriptase-Polymerase Chain Reaction  Kamesh.
A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk.

A Rapid Polymerase Chain Reaction-Based Screening Method for Identification of All Expanded Alleles of the Fragile X (FMR1) Gene in Newborn and High-Risk.
Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis  Jeanne A. Jordan, Mary Beth.

Real-Time Polymerase Chain Reaction for Detecting Bacterial DNA Directly from Blood of Neonates Being Evaluated for Sepsis  Jeanne A. Jordan, Mary Beth.
Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.

Optimization of a Relative Telomere Length Assay by Monochromatic Multiplex Real- Time Quantitative PCR on the LightCycler 480  Anthony Y.Y. Hsieh, Sara.
Measurements of Human Papillomavirus Transcripts by Real Time Quantitative Reverse Transcription-Polymerase Chain Reaction in Samples Collected for Cervical.

Measurements of Human Papillomavirus Transcripts by Real Time Quantitative Reverse Transcription-Polymerase Chain Reaction in Samples Collected for Cervical.
Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya.

Development and Evaluation of a SYBR Green–Based Real-Time Multiplex RT-PCR Assay for Simultaneous Detection and Serotyping of Dengue and Chikungunya.
Volume 61, Issue 1, Pages (January 2002)

Volume 61, Issue 1, Pages (January 2002)
MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density  Pang-Kuo Lo, Hanano Watanabe, Pi-Chun.

MethySYBR, a Novel Quantitative PCR Assay for the Dual Analysis of DNA Methylation and CpG Methylation Density  Pang-Kuo Lo, Hanano Watanabe, Pi-Chun.
High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations  Bjørnar Gilje, Reino Heikkilä, Satu.

High-Fidelity DNA Polymerase Enhances the Sensitivity of a Peptide Nucleic Acid Clamp PCR Assay for K-ras Mutations  Bjørnar Gilje, Reino Heikkilä, Satu.

Similar presentations

Ads by Google