DNA, Circular ; DNA, Viral ; Hepatitis B virus ; genetics

DNA, Circular ; isolation & purification ; DNA, Viral ; isolation & purification ; Hepatitis B ; diagnosis ; Hepatitis B virus ; genetics ; Humans

Originating but free from chromosomal DNA, extrachromosomal circular DNAs (eccDNAs) are organized in circular form and have long been found in unicellular and multicellular eukaryotes. Their biogenesis and function are poorly understood as they are characterized by sequence homology with linear DNA, for which few detection methods are available. Recent advances in high-throughput sequencing technologies have revealed that eccDNAs play crucial roles in tumor formation, evolution, and drug resistance as well as aging, genomic diversity, and other biological processes, bringing it back to the research hotspot. Several mechanisms of eccDNA formation have been proposed, including the breakage-fusion-bridge (BFB) and translocation-deletion-amplification models. Gynecologic tumors and disorders of embryonic and fetal development are major threats to human reproductive health. The roles of eccDNAs in these pathological processes have been partially elucidated since the first discovery of eccDNA in pig sperm and the double minutes in ovarian cancer ascites. The present review summarized the research history, biogenesis, and currently available detection and analytical methods for eccDNAs and clarified their functions in gynecologic tumors and reproduction. We also proposed the application of eccDNAs as drug targets and liquid biopsy markers for prenatal diagnosis and the early detection, prognosis, and treatment of gynecologic tumors. This review lays theoretical foundations for future investigations into the complex regulatory networks of eccDNAs in vital physiological and pathological processes.
Male ; Female ; Animals ; Humans ; Swine ; DNA, Circular/genetics* ; Genital Neoplasms, Female ; Semen ; DNA ; Reproduction

BACKGROUNDSuccessful treatment of hepatitis B can be achieved only if the template for hepatitis B virus (HBV) DNA replication, the covalently closed circular HBV DNA (cccDNA) can be completely cleared. To date, detecting cccDNA remains clinically challenging. The purpose of this study was to develop a nested real-time quantitative polymerase chain reaction (PCR) assay for detecting HBV cccDNA in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (MMNCs).

METHODSBased on the structural differences between HBV cccDNA and HBV relaxed circular DNA (rcDNA), two pairs of primers were synthesized as well as a downstream TaqMan probe. Blood and bone marrow samples were collected from hepatitis B patients and healthy controls. To remove rcDNA, samples were incubated with mung bean nuclease and the resultant purified HBV cccDNA was then amplified by nested real-time fluorescence quantitative PCR. The cccDNA levels were calculated using a positive standard.

RESULTSThe nested real-time fluorescence quantitative PCR method for HBV cccDNA was successful, with a linear range of 3.0 × 10(2) copies/ml to 3.9 × 10(8) copies/ml. Of the 25 PBMC samples and 7 MMNC samples obtained from chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were positive for HBV cccDNA, while all of the 21 PBMC samples from healthy controls were negative.

CONCLUSIONThe nested real-time fluorescence quantitative PCR may be used as an important tool for detecting cccDNA in hepatitis B patients.

Cells, Cultured ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Hepatitis B virus ; genetics ; Humans ; Real-Time Polymerase Chain Reaction ; methods

Animals ; DNA, Circular ; analysis ; Hepatitis B ; virology ; Hepatitis B virus ; genetics ; Humans

To verify that the circular forms of bocavirus genome exist in their host, bocavirus episomes were detected in fecal samples of healthy piglets using a semi-nested PCR method. Two species of porcine bocaviruses (PBoVG2-episome and PBoVG3-episome) were identified for the first time. The relevant terminal sequences of the noncoding region (405 and 511 nt, respectively) were also obtained. Sequence analyses and secondary structure prediction indicated that the PBoVG2-episome was more similar to that of human bocavirus 3 (HBoV3) but the PBoVG3-episome was quite different from that of other members of the genus Bocavirus. Discovery of episomal forms of porcine bocaviruses (PBoV) suggested that PBoV, like HBoV, used a different replication mechanism from other parvoviruses. The sequencing of episome Inverted Terminal Repeats (ITRs) also contributes to a possible alternative strategy for constructing infectious molecular clones of bocavirus in a future study.
Animals ; Base Sequence ; Bocavirus ; genetics ; physiology ; DNA, Circular ; genetics ; DNA, Viral ; genetics ; Genome, Viral ; genetics ; Molecular Sequence Data ; Polymerase Chain Reaction ; Swine ; virology

OBJECTIVETo establish a nested real-time quantitative polymerase chain reaction (PCR) assay for detection of hepatitis B virus covalently closed circular DNA in PBMC( peripheral blood monocyte) and MMNC (marrow monocyte).

METHODSBased on the structural differences between HBVcccDNA and HBV rcDNA, two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed. To remove rcDNA, cccDNA was processed by Mung Bean Nuclease,and then amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers. According to the standard preparation, cccDNA levels of specimen were calculated.

RESULTSWe have established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully, and the linear range is from 5.0 x 10(2) to 3. 9 x 10(7) copies per milliliter. Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients, 3 MMNC samples and 9 PBMC samples were HBV cccDNA positive, while all of the 21 healthy donator blood PBMC samples were negative.

CONCLUSIONSThe nested real-time fluorescent quantitative PCR method may be applied to detect HBVcccDNA level in PBMC and MMNC. HBVcccDNA can be detected in PBMC and MMNC.

DNA, Circular ; genetics ; DNA, Viral ; genetics ; Hepatitis B ; diagnosis ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Leukocytes, Mononuclear ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

Covalently closed circular DNA (cccDNA) is the existing form of the HBV DNA in the nucleus of host cells and also the original template of HBV replication; its long-term presence in the nucleus makes it difficult to be eliminated by current antiviral drugs; and it becomes the key factor of continuous HBV infection and relapse after antiviral suspension. Detection of HBV cccDNA is of great significance for further understanding the life cycle of HBV and providing guidance for antiviral treatment. This article aims to review the detection and its clinical significance to the advancement of researches on hepatitis B virus cccDNA.
DNA, Circular ; blood ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVETo establish a new method to detect HBV cccDNA quantitatively and to apply it to detect cccDNA in liver needle biopsy specimens of chronic hepatitis B patients.

METHODSThe sequences of HBV DNA genotypes A through G were analyzed. According to the different sequence structure of cccDNA and rcDNA, primes and probe were designed in highly conservative region outside the nick of cccDNA in order to amplify cccDNA but not rcDNA. The best conditions of this method were found after testing experiments. Also we checked its specificity and sensitivity and reproducibility. The products of PCR were sequenced in order to ascertain if it was the right region expected. To amplify with standard plasmid ranged from 10(2) to 10(10) copies/ml to measure the sensitivity and amplify in parallel with standard plasmid of 10(6) copies/ml for 30 replicates so as to measure its reproducibility. DNA was extracted from 32 needle liver biopsy specimens of chronic hepatitis B patients. The cccDNA was quantitatively detected with this method. The data of cccDNA obtained before and after therapy and their relationship with total HBV DNA were analyzed. RESULTS Results of sequencing showed that the PCR product was from the right region. The sensitivity was 10(3)-10(10) copies/ml. The Ct value was 29.69+/-0.31 and the coefficient of variability was 1.04 percent calculated from the data of 30 PCR reactions with standard plasmid. The percentage of decrease in serum HBV DNA, total HBV DNA in liver and cccDNA in liver were 0.49+/-0.17, 0.22+/-0.18 and 0.16+/-0.28 respectively. There is 47 percent-98 percent cccDNA in total HBV DNA in liver and the mean is 81.5 percent.

CONCLUSIONThe method is good because of the simple and convenient operation, the high specificity, the wide linear detection range and the fine reproducibility. Therefore it can be used for both scientific research and clinical purpose. Lamividine can significantly inhibit serum HBV DNA by, but its inhibitory effect on cccDNA in liver was rather weak.

DNA, Circular ; genetics ; DNA, Viral ; blood ; genetics ; Hepatitis B ; diagnosis ; virology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

S1 nuclease (from Aspergillus oryzae) is a specific enzyme to degrade single stranded DNA or RNA molecules. It has been reported to be able to convert superhelical circular DNA molecules into open circle or linear forms under certain conditions, but this function has not been well explored. In order to use the action of S1 nuclease to linearize circular DNA and develop a novel way of cloning microcircular DNAs, the pUC19 was used to investigate the relationship between the linearization efficiency of S1 nuclease and the amount of enzyme used. By this way the optimal conditions for linearization of circular DNAs by S1 nuclease would be determined. 0.3u to 17u S1 nuclease per 100ng pUC19 DNA was added into a 25 microL system, respectively, to perform the reaction. The effectiveness of enzyme digestion was realized by electrophoresis in a 1.2% agarose gel. The results showed that along with the increase in enzyme amount from 0.3u to 17u a gradual decrease in the superhelical form, a gradual increase in the linear form and then in the circular form was obvious. The conversion from superhelical form to linear and circular form was directly related to the enzyme amount used. A higher proportion of linear DNA molecules was achieved by using 5 to 17u S1 nuclease per 100ng DNA. Besides, electrophoretic mobility of the S1 nuclease-linearized pUC19 was the same as that of the linear form produced by restriction enzyme digestion. According to the result of phiX174 digested by S1 nuclease it has been proposed that the enzyme cleaves first randomly on one site of one strand, thus converting the superhelical molecules into open circle form, and then on the same site of the complementary strand to produce the linear form. Therefore, the S1 nuclease-linearized DNA molecules are intact in the sense of their length and can be used for cloning. The plasmid-like DNA pC3 from cucumber mitochondria is a double stranded circular DNA molecule with about 550bp and the smallest known plasmid-like DNA in eukaryotic mitochondria. Many attempts have been made to linearize the molecule by using restriction enzymes but failed. Therefore, S1 nuclease was used to linearize pC3 based on the results obtained with pUC19. The linearized pC3 DNA molecules formed a very sharp band in a 2.5% agarose gel after electrophoresis. They were then recovered from the gel, added an "A" tail and ligated with T-vector. After transformation into E. coli JM109 cells, the positive clones were, screened by the blue-white selection. The insert was then cut using restriction enzymes EcoRI and Pst I. The result of electrophoresis shows that the electrophoretic mobility of the insert is just the same as that predicted. A 32 P-labled probe was synthesized using pC3 as the template and Southern blot analysis was carried out. The result shows that the inserted DNA is hybridized to the probe, which indicates that the cloned DNA fragment is from pC3. The sequence information of the insert shows that the plasmid-like DNA pC3 was 537bp in length. The nucleotide sequence was deposited in the GenBank (the accession number is AF522195).
Blotting, Southern ; Cloning, Molecular ; methods ; DNA, Circular ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Single-Strand Specific DNA and RNA Endonucleases ; genetics ; metabolism