JANUS KINASE V617F MUTATION DETECTION IN PATIENTS WITH MYELOFIBROSIS
Nikolova D1,2,*, Yordanov A2, Damyanova V1,2, Radinov A2, Toncheva D1
*Corresponding Author: Dragomira Nikolova, Ph.D., Department of Medical Genetics, Medical Faculty, Medical University Sofia, 2 Zdrave Street, 1431 Sofia, Bulgaria. Tel: +359888-103-456, E-mail: dmb@abv.bg
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MATERIALS AND METHODS

Two hundred and thirty-two patients with different diagnoses (Figure 1) were referred for analysis to the genetic laboratory of the Clinic of Hematology, University Hospital “St.Ivan Rilski,” Sofia, Bulgaria. DNA was extracted from venous blood of patients using a commercial kit (QIAamp DNA Blood Mini Kit; Qiagen Inc., Valencia, CA, USA). The procedure was executed in accordance with the manufacturer’s recommendations. DNA samples were collected and analyzed for a total period of 3 years (from January 2016 to December 2018). All of the patients gave written informed consent for genetic analysis upon their acceptance at the Clinic. JAK2 V617F Mutation Detection by Restriction Analysis. Previously isolated DNA samples were amplified using JAK2 codon 617 mutation specific primers (V617F) (forward primer sequence: 5’-GGG TTT CCT CAG AAC GTT-3’ and reverse primer sequence: 5’-TCA TTG CTT TCC TTT TTC-3’). Amplifications were performed for 32 cycles with Taq polymerase (Qiagen Inc.), annealing temperature of 60 °C, and standard amplification conditions. Polymerase chain reaction (PCR) of the human JAK2 exon was performed as described by Baxter et al. [13]. The amplified 460 bp fragments were enzymatically digested using BsaXI restriction enzyme (New England Biolabs® Inc., Ipswich, MA, USA). Fragments of three different sizes (241, 189 and 30 bp) resulted after digestion of the wild type allele, the mutant allele remained undigested (Figure 2). Digested fragments were separated in 2.0% agarose gel. Visualization of the restriction fragments was achieved by ethidium bromide.



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