Fluorescence melting curve analysis for the detection of the bcl-1/JH translocation in mantle cell lymphoma.
PCR amplification and product analysis for the detection of chromosomal translocations such as bcl-1/JH have traditionally been performed as a two-step process with separate amplification and product detection. PCR product detection has generally entailed gel electrophoresis, hybridization, or sequencing for confirmation of assay specificity. By using a microvolume fluorimeter integrated with a thermal cycler and the PCR compatible double-stranded DNA (dsDNA) binding dye SYBR Green I, we simultaneously amplified and detected bcl-1/JH translocation products by using rapid cycle PCR and fluorescence melting curve analysis. We analyzed DNA from 25 cases of lymphoproliferative disorders comprising 12 previously documented bcl-1/JH-positive mantle cell lymphomas, and 13 reactive lymphadenopathies. The samples were coded and analyzed in a blind manner for the presence of bcl-1/JH translocations by fluorescence melting curve analysis. The results of fluorescence analysis were compared with those of conventional PCR and gel electrophoresis. All of the 12 cases (100%) previously determined to be bcl-1/JH positive by conventional PCR analysis showed a characteristic sharp decrease in fluorescence at about 86 degrees C by melting curve analysis. For easier visualization of melting temperatures (Tm), fluorescence melting peaks were obtained by plotting the negative derivative of fluorescence over temperature (-dF/dT) versus temperature (T). Dilutional assays revealed that fluorescence melting curve analysis was more sensitive than conventional PCR and agarose gel electrophoresis with ultraviolet transillumination by as much as 40-fold. Our results indicate that nucleic acid amplification integrated with fluorescence melting curve analysis is a simple, reliable, sensitive, and rapid method for the detection of bcl-1/JH translocations. The feasibility of specific PCR product detection without electrophoresis or expensive fluorescently labeled probes makes this methodology attractive for studies in molecular pathology.
Bohling
S D
SD
Department of Pathology, ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah, USA.
Wittwer
C T
CT
King
T C
TC
Elenitoba-Johnson
K S
KS
D019938:Cyclin D1 / Q000235:genetics*; D004273:DNA, Neoplasm / Q000235:genetics; D005470:Fluorometry; D015321:Gene Rearrangement / Q000235:genetics; D005914:Globins / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D006801:Humans; D007201:Indicator Dilution Techniques; D008228:Lymphoma, Non-Hodgkin / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014178:Translocation, Genetic / Q000235:genetics*
D004273:DNA, Neoplasm; D019938:Cyclin D1; D005914:Globins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases