Combined Locked Nucleic Acid and Molecular Beacon Technologies for Sensitive Detection of the JAK2V617F Somatic Single-Base Sequence Variant

摘要

We developed a quantitative and very sensitive method that combines molecular beacon (1) and locked nucleic acid (LNA) technologies (2) in a single sealed tube. We used the oncogenic somatic JAK2 V617F single-base sequence variant, observed in a broad range of Philadelphia chromosome-negative myeloproliferative diseases (MPDs) (3), to test this method of detection of somatic point variants. We used the molecular beacon to specifically detect the JAK2 V617F mutant allele and the LNA oligonucleotide to limit amplification of the wild-type JAK2 sequences (see Fig. 1 in the Data Supplement that accompanies the online version of this letter at http://www.clinchem.org/content/vol52/issue7). With this method, we detected very small quantities of mutant alleles in tubes containing the homozygous JAK2 V617F mutant cell line (HEL cell line) mixed with various amounts of wild-type cells and in clinical specimens containing small amounts of JAK2 V617F mutated cells.Primers and beacon probe were designed with the freeware MELT-CALC, Ver. 2.0 (http://www.meltcalc.de/). The melting temperature ( T m) of the molecular beacon stems was calculated by use of the Mfold algorithm (http://www.bioinfo.rpi.edu/applications/mfold/old/dna/form1.cgi), whereas the T m of the LNA was calculated via the Exiqon website (http://lna-tm.com). …