Comparison of Sanger sequencing and next generation sequencing methods for investigation of JAK2 Exon 12 mutations in follow-up of patients with chronic myeloproliferative disease and JAK2 V617F non-mutation

Abstract

Myeloproliferative neoplasms (MPNs) are clonal disorders of hematopoietic stem cells with increased proliferation and efficient maturation of myeloid cells, leading to peripheral blood leukocytosis and excess erythrocytes or platelets. Mutations of the JAK2 V617F, CALR and MPL genes confirm the diagnosis of myeloproliferative neoplasm (MPN). Mutations in JAK2 have been identified in the majority of patients with PV, ET and PM, highlighting the importance of constitutive activation of JAK2 signaling induced by mutations. In our study, Sanger Sequencing and Next Generation Sequencing methods were used to search for JAK2 Exon 12 mutations in 100 individuals who suffered from Chronic Myeloproliferative Disease and did not have JAK2 V617F mutation by Real-Time PCR method, and the results were examined comparatively. The examination was made with DNA material isolated from peripheral blood samples taken from patients who were referred to Ankara Numune Training and Research Hospital (ANEAH) Genetic Diseases Diagnosis Center. First of all, individuals who have with negative JAK2 V617F RT-PCR test results were selected. PCR was performed by adjusting sufficient amounts and concentrations from the DNA samples obtained from the peripheral blood of these patients. After the PCR process, the JAK2 Exon 12 regions were sequenced and examined using the Sanger sequencing method. A Next Generation Sequencing (NGS) study was performed by creating libraries from the DNA of the patients whose JAK2 Exon 12 region was negative, and the results were analyzed using the database. Some of the studies were conducted at the ANEAH Genetic Diseases Diagnostic Center, and the other 46 patients were performed at the Intergen Genetic Diseases Diagnosis Center within the scope of NGS study service procurement. According to the analyzes made, the results of Sanger Sequencing and Next Generation Sequencing studies showed similarity. Despite the deep bottom readings, a different result could not be obtained from the Sanger Sequencing method in the NGS study.

Keywords

• Chronic myeloproliferative disease
• Sanger sequencing
• next generation sequencing
• Janus Kinas 2
• Real-Time PCR

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