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

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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 33-42 (January 2008) DOI: 10.2353/jmoldx.2008.070076 Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 0.997 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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

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 0.996 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 2008 10, 33-42DOI: (10.2353/jmoldx.2008.070076) Copyright © 2008 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions