(B) Physical map of our standard plasmids. The arrow heads show the primers which amplify the reference control sequence. The asterisk indicates the JAK2V617F mutation position (cDNA 1849 G>T). (A) Physical map of JAK2 exon 14-22, including the mutation position on exon 14 and the 264-bp reference control region on exon 21. The reference sequence, plasmid construction, and validation of the accuracy of artificial plasmid mixtures as quantification standards. Other relevant information is also available in the Online Supplementary Appendix. The sequences and working concentrations of all primers, probes and the blocker are listed in Online Supplementary Table S1. With these refinements, we designated our novel method as quantitative competitive allele-specific TaqMan Duplex PCR (qCAST-Duplex PCR) assay. It competed with the mutant allele-specific primer and preferentially annealed to WT templates with high affinity, which impeded their non-specific amplification. Lastly, but most importantly, we designed an oligonucleotide (ON) WT template blocker containing a di-deoxycytidine at its 3′ end (3′-ddCTP). The latter could be a major but unexpected benefit when some rare samples of limited quantity are examined. The designs allowed us to perform duplex PCR (including mutant-specific assay and copy-number-normalizing control assay) in one tube and quantify two PCR products concurrently, which also helped minimize sampling bias and decrease the consumption of genomic DNA. Thirdly, allele-specific (AS) primers and a FAM-containing TaqMan probe (Bio-Rad, USA) were used for JAK2V617F detection, whereas primers for the control gene were mixed with a HEX-containing probe to quantify JAK2 exon 21 ( Figure 1C), and the calculation of mutant AB was determined by the ΔCt method. They were used to establish quantification standards. The plasmid pair had the same size (3306 bps) and could be easily manipulated with identical copy numbers. In the second step, the exon 21 control sequence, along with either one of the target regions, were cloned into a yT&A cloning vector (Yeastern Biotech, Taiwan) to generate two standard plasmids ( JAK2_WT_Ctrl_yT&A and JAK2_V617F_Ctrl_yT&A, representing 0% and 100% mutant AB, respectively) ( Figure 1B). It improved the efficacy of copy number normalization by directly calibrating equal copies of target DNA with the reference control within the same gene. Firstly, we decided to select JAK2 exon 21 as our reference control ( Figure 1A). To improve the accuracy of JAK2V617F detection and quantification, we made some refinements to the allele-specific real-time polymerase chain reaction (qPCR) assay and created a novel method. 1 Although DNA from JAK2-mutated UKE-1 and HEL cells are commonly used for this purpose, 2, 3 none of these cells are considered ideal, as the HEL cells carry multiple copies of JAK2, and UKE-1 cells do undergo clonal evolution with increasing JAK2 copies during in vitro cultures. 1 The study also concluded by delineating the importance of using well-defined, accurate standards to refine JAK2 quantitative assays. Unfortunately, there were significant discrepancies in the allele burden (AB) quantification when blinded samples were tested in a multicenter study. Since its discovery, many diagnostic techniques have been applied in its detection.
#Been qcast driver
JAK2V617F mutation is one of the key driver mutations in myeloproliferative neoplasms (MPN).
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