Quantification of bcl-2/JH fusion sequences and a control gene by multiplex real-time PCR coupled with automated amplicon sizing by capillary electrophoresis.
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.
Sanchez-Vega
Beatriz
B
Division of Pathology and Laboratory Medicine, The University of Texas MD Anderson Cancer Center, Houston 77030-4095, Texas, USA.
Vega
Francisco
F
Medeiros
L Jeffrey
LJ
Lee
Ming S
MS
Luthra
Rajyalakshmi
R
D002883:Chromosomes, Human, Pair 14 / Q000235:genetics*; D002887:Chromosomes, Human, Pair 18 / Q000235:genetics*; D017931:DNA Primers / Q000737:chemistry; D004273:DNA, Neoplasm / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D007133:Immunoglobulin Joining Region / Q000235:genetics; D008224:Lymphoma, Follicular / Q000235:genetics* / Q000473:pathology; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics*; D012680:Sensitivity and Specificity; D014178:Translocation, Genetic
D017931:DNA Primers; D004273:DNA, Neoplasm; D007133:Immunoglobulin Joining Region; D015514:Oncogene Proteins, Fusion; D019253:Proto-Oncogene Proteins c-bcl-2