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The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay  Ralph Stadhouders, Suzan D.

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Presentation on theme: "The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay  Ralph Stadhouders, Suzan D."— Presentation transcript:

1 The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease Assay  Ralph Stadhouders, Suzan D. Pas, Jeer Anber, Jolanda Voermans, Ted H.M. Mes, Martin Schutten  The Journal of Molecular Diagnostics  Volume 12, Issue 1, Pages (January 2010) DOI: /jmoldx Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

2 Figure 1 Oligonucleotides used to study the effects of 3′ end primer-template mismatches on nucleic acid detection and quantification with real-time Taqman PCR. Panel (A) represents a linear map of the HIV-HMPV insert, in which the location of both viral DNA sequences and corresponding primers and probes are depicted. Panel (B) shows the nucleotide sequences of the primers (black) as located in the vector. Arrows indicate the positions on the HIV-hMPVpGA4 vector that have been subjected to site-directed mutagenesis. fwd = forward primer, rev = reverse primer, PRB = probe. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

3 Figure 2 Effects of primer-template mismatches on the quantification of nucleic acids using real-time Taqman PCR. Each panel represents the effects of one single mismatch (depicted as a primer-template mismatch) at different positions on the HIV-hMPVpGA4 construct. The position of the mismatch and the primer in which it is located are shown on the x axis, while the y axis shows the average increase in detected Ct-value as compared with the complementary situation. Amplifications were performed using the 2× Taqman Universal PCR Mastermix. Error bars represent the SD (n = 4). Differences in Ct-values were tested for statistical significance using a Student t-test. Significant values (*P < 0.05) are indicated nt = nucleotide, rev = reverse primer, fwd = forward primer. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

4 Figure 3 Effects of primer-template mismatches on the quantification of nucleic acids using a Taq/MMLV-based real-time Taqman RT-PCR. Each panel represents the effects of the 12 individual mismatches (depicted as primer-template mismatches) per primer. The type and position of the individual mismatches are shown on the x axis, while the y axis shows the average increase in detected Ct-value as compared with the complementary situation. Amplifications were performed using the Taqman PCR Core Reagents Kit, combined with Multiscribe Reverse Transcriptase and RNase inhibitors. Error bars represent the SD (n = 4). Differences in Ct-values were tested for statistical significance using a Student t-test. Significant values (*P < 0.05) are indicated nt = nucleotide, REV = reverse primer, FWD = forward primer. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

5 Figure 4 Effects of primer-template mismatches on the quantification of nucleic acids using rTth DNA polymerase-based real-time Taqman RT-PCR. Each panel represents the effects of the 12 individual mismatches (depicted as primer-template mismatches) per primer. The type and position of the individual mismatches are shown on the x axis, while the y axis shows the average increase in detected Ct-value as compared with the complementary situation. Amplifications were performed using the Taqman EZ RT-PCR Kit. Error bars represent the SD (n = 4). Differences in Ct-values were tested for statistical significance using a Student t-test. Significant values (P < 0.05) are indicated by an asterisk. nt = nucleotide, REV = reverse primer, FWD = forward primer. The Journal of Molecular Diagnostics  , DOI: ( /jmoldx ) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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