Detecting hbe gene using dna extracted from urine sediments by chelex-plus-heating technique

• Main Authors: Areerat Pankham, Thanusak Tatu
• Format: Journal
• Published: 2020
• Subjects: Biochemistry, Genetics and Molecular Biology
• Online Access: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=85091600930&origin=inward; http://cmuir.cmu.ac.th/jspui/handle/6653943832/70170
• Institution: Chiang Mai University

© 2020 ABRF. Detecting βE-allele or hemoglobin E (HbE) gene by PCR generally uses DNA prepared from blood leukocytes. However, blood drawing is invasive and prone to injury and infection. The so-called Chelex-plus-heating protocol for DNA extraction from urine sediments was performed in this study. In this protocol, urine sediments were incubated at 37°C with Chelex-100 resin, followed by heating in boiling water for 20 min, and were spun for 1 min to harvest the DNA-containing supernatant. The obtained DNA was subsequently used in amplification refractory mutation system (ARMS)-PCR for detecting βE-allele. The ARMS-PCR results obtained from urine-DNA were compared to those produced by ARMS-PCR using blood-leukocyte DNA. It was found that the Chelex-plus-heating technique successfully released DNA of good quality with sufficient quantity from urine sediments. Twenty microliters of urine having ~1111 cells/ml was sufficient to provide good-quality DNA for PCR reaction for HbE genotyping by ARMS-PCR. It was concluded that the Chelex-plus-heating technique was suitable for preparing the DNA from urine sediments. Being simple and less costly, this technique should promote effective control of HbE for countries having a limited budget.