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Updated: Jun 18, 2025
High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue
Published on: February 1, 2011
Selective Ligase-Based Sample Processing-Free Discrimination and Detection of Site-Specific DNA
Jiahui Zhao1, Jingli Yan1, Jing Li1
1Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province (Project Number: 22567620H), State Key Laboratory of New Pharmaceutical Preparations and Excipients, College of Chemistry and Materials Science, Hebei University, Baoding 071002, China.
Abstract:
Accurate detection of site-specific 5-hydroxymethylcytosine (5hmC) in genomic DNA is of great significance, but it is technically challenging to directly distinguish very low levels of 5hmC from their abundant cytosine/5-methylcytosine (C/5mC) analogues. Herein, we wish to propose a selective ligase-mediated mechanism (SLim) that can directly discriminate 5hmC from C/5mC with a high specificity without the use of any sample processing protocol. In this new design, we discovered that HiFi Taq DNA Ligase can well tolerate the mismatched 5hmC/A base-pairing and then effectively ligate the associated nicking site while the mismatched 5mC/A or C/A pairs cannot be recognized by HiFi Taq DNA Ligase, providing a new way for direct and selective discriminating 5hmC from its similar analogues. Ultrasensitive and selective quantification of site-specific 5hmC is realized by coupling the SLim with polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).
