|
|
|
| Comparison of two methods for extracting hepatitis B virus covalently closed circular DNA |
| HU Kongying, JIN Yutao, YE Jianyu, ZAI Wenjing, WANG Yang, LI Yumeng, CHEN Jieliang |
| Department of Medical Microbiology and Parasitology, MOE & NHC Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China |
|
|
|
|
Abstract Hepatitis B virus (HBV) is a hepatotropic DNA virus that can deposit a covalently closed circular DNA form (cccDNA) in the nucleus of infected hepatocytes, which serves as a reservoir for HBV genome and the template for transcribing HBV RNAs. The persistence of cccDNA is the key of HBV chronicity and the main obstacle for HBV cure, and thus cccDNA has always been a focus of HBV research. The efficient extraction of cccDNA from cells is required for accurate detection and quantification. Hirt DNA extraction is a method for isolation of the extrachromosomal DNA in eukaryotes and has been applied for cccDNA extraction. However, the procedure of Hirt DNA extraction is laborious and time-consuming. To simplify the procedure, there has been reports of ”modified Hirt DNA extraction” combined with silicon-based resins column for rapid extraction of extrachromosomal DNA. However, the difference of extraction efficacy between the two methods remains unknown. Here, the two methods were parallelly compared by using Southern Blot and qPCR in various cell culture-based HBV transfection, replication and infection systems. The results suggested that “the modified Hirt-Silica Gel Membrane method” has the potential to enable a fast HBV cccDNA extraction with equally extraction efficacy and specificity compared with “the original Hirt-Phenol/Chloroform method”．
|
|
Received: 17 December 2020
Published: 25 October 2021
|
|
|
|
Corresponding Authors:
CHEN Jieliang
E-mail: jieliangchen@fudan.edu.cn
|
|
|
|
|
|
|
2021, Vol. 16 
