The use of fluorescent molecular beacons in real time PCR of IgH gene rearrangements for quantitative evaluation of multiple myeloma.
BACKGROUND AND OBJECTIVES Fluorescent molecular beacons have been employed as hybridization probes in real time quantitative PCR to quantify residual disease in multiple myeloma (MM).


METHODS
After clinical diagnosis of MM, the CDR1, CDR2 and CDR3 regions of the IgH gene were analysed and sequenced to identify its clonal nature. Unique sequences of the clonal IgH rearrangement were used to design specific molecular beacon probes for each MM patient. A molecular beacon probe for the beta-globin gene was used as a reference control to calculate relative amounts of the clonal B-cell population.


RESULTS
Optimization of probe design resulted in the use of a competitive sequence at the IgH area target between the loop and part of the stem of the molecular beacon. Cycling conditions and fluorescence temperature acquisition were optimized for a Light Cycler. To validate this method for the follow-up of treated MM patients, we investigated accuracy, as well as interassay and intrassay reproducibility.


CONCLUSIONS
Our results indicated that real time PCR with specific molecular beacons provides a feasible, accurate and reproducible method for the determination of minimal residual disease in MM.
Martínez-López
J
J
Servicio de Hematología, Hospital Universitario 12 de Octubre, Madrid, Spain. jmartinezlo@yahoo.es
Lahuerta
J J
JJ
Salama
P
P
Ayala
R
R
Bautista
J M
JM
D015342:DNA Probes / Q000235:genetics; D005069:Evaluation Studies as Topic; D005456:Fluorescent Dyes / Q000737:chemistry; D015321:Gene Rearrangement / Q000235:genetics; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics*; D005803:Genes, Immunoglobulin / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007133:Immunoglobulin Joining Region / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D009101:Multiple Myeloma / Q000235:genetics*; D018365:Neoplasm, Residual / Q000235:genetics; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D010954:Plasmacytoma / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D015342:DNA Probes; D005456:Fluorescent Dyes; C115494:IgH-MMSET fusion protein, human; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D015514:Oncogene Proteins, Fusion