Application of Self-Quenched JH Consensus Primers for Real-Time Quantitative PCR of IGH Gene to Minimal Residual Disease Evaluation in Multiple Myeloma

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Application of Self-Quenched JH Consensus Primers for Real-Time Quantitative PCR of IGH Gene to Minimal Residual Disease Evaluation in Multiple Myeloma Joaquin Martinez-Lopez, Pilar Martínez-Sanchez, Ramon Garcia-Sanz, Maria Eugenia Sarasquete, Rosa Ayala, Marcos Gonzalez, Jose Manuel Bautista, David Gonzalez, Jesus San Miguel, Guillermo Garcia-Effron, Juan Jose Lahuerta The Journal of Molecular Diagnostics Volume 8, Issue 3, Pages 364-370 (July 2006) DOI: 10.2353/jmoldx.2006.050101 Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Patient rearrangements and oligonucleotide sequences. Junction regions VDH, DHJ, or both are indicated with vertical arrows. N nucleotide insertions and somatic mutations are represented in lowercase letters. Allele-specific and JH intronic primer are underlined with dotted lines, probe sequences are underlined with black lines, and the self-quenched sequence is inside the rectangle. All cases have been sequenced using the JH consensus primer. Cases 10160, 9985, and 4526 (cases that did not work) have also been sequenced using the next JH intronic primer just downstream to study the mutations present in the probe and primer binding sites. The Journal of Molecular Diagnostics 2006 8, 364-370DOI: (10.2353/jmoldx.2006.050101) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Scheme and standard curves of two methodologies. A: Example of TaqMan PCR. Amplification curves of one patient's diagnostic sample, several dilutions to calculate standard curve, the sample after treatment (MRD), and the sample at the time of the relapse. NTC, nontemplate control. B: Example of self-quenched PCR. C: Melting curves in one self-quenched method case. There are two different melting temperatures: 84°C for the specific fragments (B) and 77°C for the nonspecific fluorescence (A). The Journal of Molecular Diagnostics 2006 8, 364-370DOI: (10.2353/jmoldx.2006.050101) Copyright © 2006 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions