title: Detection of Janus Kinase 2 gene single point mutation in real samples with electrochemical DNA biosensor.
abstract: Janus Kinase 2 (JAK2) gene single point mutations, which have been reported to be associated with myeloproliferative disorders, are usually detected through conventional methods such as melting curve assays, allele-specific and quantitative Polymerase Chain Reactions (PCRs). Herein, an electrochemical biosensor for the detection of a Guanine (G) to Thymine (T) transversion at nucleotide position 1849 of the JAK2 gene was reported. Due to clinical importance of this mutation, easy and sensitive tests are needed to be developed. Our aim was to design a biosensor system that is capable of detecting the mutation within less than 1h with high sensitivity. For these purposes, an electrochemical sensing system was developed based on detecting hybridization. Hybridization between probe and its target and discrimination of single point mutation was investigated by monitoring guanine oxidation signals observed at +1.0 V with Differential Pulse Voltammetry (DPV) by using synthetic oligonucleotides and Polymerase Chain Reaction (PCR) amplicons. Hybridization between probe and PCR amplicons was also determined with Electrochemical Impedance Spectroscopy (EIS). We successfully detect hybridization first in synthetic samples, and ultimately in real samples involving blood samples from patients as well as additional healthy controls. The limit of detection (S/N=3) was calculated as 44 pmol of target sequence in a 40-μl reaction volume in real samples.
authors: Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, Izmir, Turkey. Electronic address: sedanur6@gmail.com.
authors: Seda Nur
authors: SN
authors: Topkaya
authors: Department of Medical Biology, Faculty of Medicine, Ege University, Izmir, Turkey.
authors: Buket
authors: B
authors: Kosova
authors: Department of Electrical and Electronic Engineering, Faculty of Engineering and Architecture, Gediz University, Izmir, Turkey.
authors: Mehmet
authors: M
authors: Ozsoz
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D004563:Electrochemistry; D006801:Humans; D053614:Janus Kinase 2 / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D017354:Point Mutation
chemical_list: D004247:DNA; D053614:Janus Kinase 2
keywords: Differential Pulse Voltammetry (DPV); Electrochemical DNA biosensor; Electrochemical Impedance Spectroscopy (EIS); JAK2V617F mutation; Single point mutation