Quantification of bcl-2/JH fusion sequences and a control gene by multiplex real-time PCR coupled with automated amplicon sizing by capillary electrophoresis

J Mol Diagn. 2002 Nov;4(4):223-9. doi: 10.1016/S1525-1578(10)60707-6.

Abstract

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.

MeSH terms

  • 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*
  • Sensitivity and Specificity
  • Translocation, Genetic

Substances

  • DNA Primers
  • DNA, Neoplasm
  • Immunoglobulin Joining Region
  • Oncogene Proteins, Fusion
  • Proto-Oncogene Proteins c-bcl-2