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

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

Beatriz Sanchez-Vega et al. J Mol Diagn. 2002 Nov.

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.

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Figures

Figure 1.

Figure 1.

Standard curves showing the initial DNA quantity versus threshold cycle (Ct). The standard curves were generated by three different real-time PCR assay conditions using serial dilutions of DNA from t(14;18)-positive cell lines: blue, mbr or mcr alone; red, co-amplification of mbr or mcr with cyclophilin; green, co-amplification of mbr or mcr with cyclophilin and using a NED-labeled JH primer.

Figure 2.

Figure 2.

Correlation between the results of multiplex real-time PCR for two _bcl-2 mbr/JH_-positive cell lines with fusion sequences of 87 bp and 220 bp. There was an almost perfect correlation (r = 0.999) between the Ct values using both cell lines at the same concentrations indicating that the efficiency of the multiplex PCR was not altered by amplicon size. Each experiment was repeated three times and each sample was analyzed in duplicate.

Figure 3.

Figure 3.

Correlation between multiplex and separate real-time PCR results for 33 _bcl-2 mbr/JH_-positive patient samples.

Figure 4.

Figure 4.

Patient 19 with four sequential bone marrow specimens. A: Amplification plots of real-time PCR assay for bcl-2 mbr/JH fusion sequences. B: Capillary electrophoresis electropherograms for each sample in A. C: Amplification plots of real-time PCR assay for cyclophilin. In A, note that a lower fluorescent signal was observed in the last bone marrow sample obtained (green) compared with the other specimens (yellow plot at time of diagnosis; red plot after 6 months, and blue plot after 13 months). As shown in B, the same size bcl-2 mbr/JH fusion sequence of 108 bp was obtained in all assays.

Figure 5.

Figure 5.

Capillary electrophoresis electropherograms showing the size of the bcl-2/JH fusion sequences obtained from one patient (patient 5) and a t(14;18)-positive cell line. bcl-2/JH fusion sequences of 154 bp and 87 bp were obtained, respectively.

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