TY - JOUR
T1 - Quantification of bcl-2/JH fusion sequences and a control gene by multiplex real-time PCR coupled with automated amplicon sizing by capillary electrophoresis
AU - Sanchez-Vega, Beatriz
AU - Vega, Francisco
AU - Medeiros, L. Jeffrey
AU - Lee, Ming S.
AU - Luthra, Rajyalakshmi
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2002/11
Y1 - 2002/11
N2 - 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.
AB - 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.
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U2 - 10.1016/S1525-1578(10)60707-6
DO - 10.1016/S1525-1578(10)60707-6
M3 - Article
C2 - 12411590
AN - SCOPUS:0036862150
SN - 1525-1578
VL - 4
SP - 223
EP - 229
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 4
ER -