A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

Table 1

Primers used for snapback primer assay, amplification refractory mutation system, TaqMan  probe qPCR, and Sanger sequencing.

	Primer	Sequence (5′-3′) and fluorescent label
Snapback	JAK2 V617F snapback	GG GAGTATGTgTCTGTGGAGACTGACACCTAGCTGTGATCCTG*
	JAK2 V617F limiting	TGAAGCAGCAAGTATGATGAG
	MPL W515L/K snapback	AC CTGCTGAGGtggCAGTTTCCTGGGGTCACAGAGCGAACCAA*
	MPL W515L/K limiting	AGCCTGGATCTCCTTGGTGAC
ARMS	JAK2 V617F outer forward	TCCTCAGAACGTTGATGGCAG
	JAK2 V617F outer reverse	ATTGCTTTCCTTTTTCACAAGAT
	JAK2 V617F specific wild-type	GCTTTGGTTTTAAATTATGGAGTATATG
	JAK2 V617F specific mutant	GTTTTACTTACTCTCGTCTCCACAAAA
	MPL W515L/K outer forward	GCCTGGATCTCCTTGGTGAC
	MPL W515L/K outer reverse	GAGGTGACGTGCAGGAAGTG
	MPL W515L/K specific wild-type	CTGTAGTGTGCAGGAAACTGTCA
	MPL W515L specific	GCCTGCTGCTGCTGAGATT
	MPL W515K specific	GCCTGCTGCTGCTGAGTAA
TaqMan	JAK2 forward primer	AAGCTTTCTCACAAGCATTTGGTTT
	JAK2 reverse primer	AGAAAGGCATTAGAAAGCCTGTAGTT
	JAK2 wild-type probe	VIC-TCTCCACAGAcACATAC-BHQ1
	JAK2 V617F probe	FAM-TCCACAGAaACATAC-BHQ1
	MPL forward primer	TAGCCTGGATCTCCTTGGTG
	MPL reverse primer	ACAGAGCGAACCAAGAATGC
	MPL wild-type probe	FAM-CTGCTGAGGtggCAGTTTC-BHQ1
	MPL W515L probe	FAM-CTGCTGAGGttgCAGTTTC-BHQ1
	MPL W515K probe	FAM-TGCTGCTGAGGaagCAGTTTCC-BHQ1
Sequencing	JAK2 V617F seq. forward	CAAAGCACATTGTATCCTCA
	JAK2 V617F seq. reverse	AGTCCTACAGTGTTTTCAGT
	MPLW515L/K seq. forward	AGCCTGGATCTCCTTGGTGACCG
	MPLW515L/K seq. reverse	TCACAGAGCGAACCAAGAATGCC

∗For the snapback primer and TaqMan probe sequences, the italic uppercases stand for the 5′-mismatch of snapback probe; the snapback probe regions are underlined; the uppercases indicate the annealing primer; and the lowercases indicate mutation sites.