Quantification of bcl-2/JH Fusion Sequences and a Control Gene by Multiplex Real-Time PCR Coupled with Automated Amplicon Sizing by Capillary Electrophoresis

• Published in: The Journal of molecular diagnostics : JMD Vol. 4; no. 4; pp. 223 - 229
• Main Authors: Sanchez-Vega, Beatriz, Vega, Francisco, Medeiros, L. Jeffrey, Lee, Ming S., Luthra, Rajyalakshmi
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2002; ASIP; American Society for Investigative Pathology
• Subjects: Chromosomes, Human, Pair 14 - genetics; Chromosomes, Human, Pair 18 - genetics; DNA Primers - chemistry; DNA, Neoplasm - genetics; Electrophoresis, Capillary - methods; HL-60 Cells; Humans; Immunoglobulin Joining Region - genetics; Lymphoma, Follicular - genetics; Lymphoma, Follicular - pathology; Oncogene Proteins, Fusion - genetics; Polymerase Chain Reaction - methods; Proto-Oncogene Proteins c-bcl-2 - genetics; Regular; Sensitivity and Specificity; Translocation, Genetic
• ISSN: 1525-1578; 1943-7811
• DOI: 10.1016/S1525-1578(10)60707-6

Follicular lymphoma is characterized by the presence of the t(14;18)(q32;q21) chromosomal translocation which juxtaposes the bcl-2 gene at 18q21 with the immunoglobulin heavy chain locus at 14q32. Quantification of t(14;18) carrying cells in FL patients can be achieved by real-time PCR, a highly sensitive technique for evaluating treatment efficacy and minimal residual disease. Despite the many advantages of real-time technology for this purpose, one disadvantage is that current real-time t(14;18) PCR assays amplify a control gene as a normalizer in a separate reaction. Since each PCR reaction has its own kinetics, separate PCR assays for target and control sequences can potentially result in inaccurate quantification of t(14;18)-positive cells. In addition, the real-time t(14;18) PCR assays do not determine the size of the amplified fusion sequence, which is helpful for excluding contamination and is commonly used to demonstrate clonal identity between pre- and post-treatment specimens from a patient. To address these limitations, we designed a multiplex real-time PCR protocol that allows amplification of control and target genes in the same reaction and precise size determination of bcl-2/JH fusion sequences by capillary electrophoresis. This multiplex PCR assay is equally sensitive to previous assays, allows more accurate quantification of bcl-2/JH fusion sequences, and is more convenient.