Polymerase Chain Reaction Analysis of t(14;18) Junctional Regions in B-Cell Lymphomas

Abstract

The polymerase chain reaction (PCR) procedure was used for rapid and highly specific amplification of the t(14;18) bcl-2/JH DNA junctional regions in B-cell lymphomas. By using Taq-polymerase and relatively long oligonucleotide primers-a 33-mer for bcl-2 and an universal 25-mer complementary to the JH consensus sequence-the primer annealing and primer extension steps could be carried out at the same temperature (70°C), thus markedly reducing the reaction time and significantly improving the specificity of the reaction. The specificity of the amplification allowed visual identification of the bcl-2/JH PCR-products in ethidium bromide stained agarose gels. DNA-sequence analysis of PCR-amplified, previously uncharacterized t(14; 18) junctional regions, confirmed the specificity of this assay. Moreover, preliminary data show that the procedure is capable of documenting the presence of occult lymphoma cells in both the peripheral blood and bone marrow.

Demonstration of Array-Based Analysis for Highly Multiplexed PCR Assays

“…The MPAD approach is similar to early PCR assays for the identification of IGH@-BCL2 translocations, in which the appropriate PCR product was identified by Southern blots that were sequentially probed with labeled IGH@ and BCL2 sequences. [43][44][45][46][53][54][55][56][57] However, the MPAD process is significantly streamlined: with prepared arrays, the MPAD protocol from PCR through visualization of results can be accomplished in a single day. We also anticipate that membrane construction can be readily mechanized for commercial preparation.…”

“…13 The sensitivity of this approach is limited compared with FISH: BIOMED-2 assays identified IGH@-BCL2 translocation in 60% to 70% using fresh or frozen FL samples, whereas outcomes on matched FFPE samples were decreased by more than a third. 6,27,28 Spurred by the limitations of BIOMED-2 and other similar assays, several groups have described alternative PCR-based assays for the IGH@-BCL2 translocation using "long distance," 16,25,29 -31 "realtime," 15,[32][33][34][35][36][37][38][39][40][41] "ELISA" like, 42 and "nested" [43][44][45][46][47] methods. The complexity and/or requirements for specialized equipment appear to have limited the widespread clinical implementation of these assays.…”

Applications of consensus polymerase chain reaction with subsequent electrophoretic distinction of amplificates

“…The present SSCP method might serve to circumvent the molecular cloning step in the mentioned protocol [ 121, since reamplifying SSCP bands after excising them from silver-stained PhastGels should provide a sufficient purification of clonal sequences. The most straightforward approach for analyzing minimal residual disease consists in obtaining sufficient sequence information for designing clonespecific PCR primers from consensus PCR products directly [13]. Despite the rapid progress in direct sequencing some amplified sequences are not readily accessible for this method.…”

“…The sequences of CDRIII forward and reverse primers were 5' CTG TCG ACA CGG CCG TGT ATT ACT G 3' and 5' AAC TGC AGA GGA GAC GGT GAC C 3', respectively [12]. To amplify IgH-bcl2 fusions, the primers 5' GAC CTT GTT TCT TGA AGG TTT CCT CGT CCC T 3' and 5' GGT GAC CAG GGT TCA CTG GCC CCA G 3' were used, which match with bc12 sequences of chromosome 18 and JH sequences of chromosome 14, respectively [13]. Usually two rounds of PCR were performed, each consisting of 25 cycles, in the case of IGH-bcl2 fusions using the nested bcl2 primer 5' GCA ATT CCG CAT TTA ATT CAT GGT ATT CAG GAT 3'.…”