Peptide nucleic acid probe for multiplex detection of BCR/ABL negative myeloproliferative neoplasm-associated gene mutations

A method for multiplex detection of a BCR/ABL negative myeloproliferative neoplasm-associated gene mutation, the method comprising:

• hybridizing a set of amplification forward and reverse primers comprising the sequences of SEQ ID NOS:
• 1 to 6, wherein each primer complementarily binds to a BCR/ABL negative myeloproliferative neoplasm-associated gene to specifically amplify a region of a mutation site, to a target DNA sample,
• amplifying a product obtained from the hybridizing step by PCR,
• adding a set of PNA probes to the amplified product, wherein each probe consists of the sequences of:
• SEQ ID NO:
• 7 binding to a mutation region of a Janus Tyrosine Kinase 2 (JAK2) gene;
• SEQ ID NO:
• 8 binding to a mutation region of a Myeloproliferative leukemia protein (MPL) gene;
• SEQ ID NO:
• 9 binding to a deletion region of a Calreticulin (CALR) Gene;
• and
• SEQ ID NO:
• 10 binding to an insertion region of the CALR Gene;
• respectively, wherein the peptide nucleic acid probe optionally contains a reporter molecule and a quencher molecule at each end of the probe,
• obtaining a melting curve;
• and
• comparing said melting curve with the standard melting temperatures of mutant genes,
• wherein the BCR/ABL negative myeloproliferative neoplasm-associated gene mutation comprises one or more selected from the group consisting of (i) a substitution of valine at position 617 of the JAK2 gene with phenylalanine (JAK2 V617F), (ii) a substitution of tryptophan at position 515 of the MPL gene with leucine (MPL W515L), (iii) a substitution of tryptophan at position 515 of the MPL gene with lysine (MPL W515K), (iv) a substitution of tryptophan at position 515 of the MPL gene with arginine (MPL W515R), (v) a substitution of tryptophan at position 515 of the MPL gene with alanine (MPL W515A), (vi) a substitution of tryptophan at position 515 of the MPL gene with serine (MPL W515S), (vii) a c.1092_1143del52 base deletion of the CALR gene (L367fs*46) and (viii) a c.1154_1155insTTGTC base insertion of the CALR gene (K385fs*47).