Rolling circle amplification is a rapid, sensitive and isothermal single-stranded DNA amplification technique that can be used with staining or probes to amplify the detection signal. This technology has been widely used in biological detection and other aspects. The present paper introduces how to design rolling circle amplification, summarize its application in the detection of pathogens, nucleic acid tumor markers, proteins, biological small biomolecules, and viruses in recent years and prospects for future development.
DNA, Single-Stranded ; Nucleic Acid Amplification Techniques

OBJECTIVE: Human papillomavirus (HPV) has been identified in the majority of invasive cervical cancer patient and has been found to contribute in a significant way to the genesis of human cervical cancer. HPV has two transforming genes that encode the oncoproteins E6 and E7, E6 can form complexes with p53 and promote p53 degradation, E7 inhibit retinoblastoma protein (RB). The p53 protein is as a phosphoprotein which co-immunoprecipitated with the SV40 T-Antigen. The wild type p53 protein is capable of suppressing the tumorigenic phenotype and regulating cell cycle. Adeno-associated virus(AAV) is a linear single stranded DNA parvovirus which is dependent upon cotransfection by a second unrelated virus to undergo productive infection. It has been well documented that AAV DNA integrates into cellular DNA as one to several tandem copies joined to cellular DNA through the termini. In order to introduce wild type p53 through AAV virus into a cervical cancer patient for gene therapy, we had constructed recombinant p53 adeno associated virus plasmid (pAAVCMVp53). METHODS: pAAVCMVp53 was created new AAV-vector system, pRc/CMVp53 including p53 cDNA and AAV-derivative vector, pASPA-AAV-CMV-polyA were made to HindIII/blunt fragments. Eluated 1.8 kb fragment of wild type p53 cDNA was ligated to pAAV-CMV-polyA, 4.9 kb fragment deprived hASPA cDNA. RESULT: Recombinant AAVCMVp53 was constructed by using pRc/CMVp53 andpASPA-AAV-CMV-polyA. This pAAVCMVp53 was confirmed by various restriction enzyme-digestions and Southern-blotting. This new vector system will be studied on expression, stability in cervical cancer cell lines and animals. CONCLUSION: This system will be one of the useful vector system for cervical cancer gene therapy.
Animals ; Antigens, Viral, Tumor ; Cell Cycle ; Cell Line ; Clone Cells ; DNA ; DNA, Complementary ; DNA, Single-Stranded ; Genetic Therapy* ; Humans ; Oncogene Proteins ; Oncogenes ; Parvovirus ; Phenotype ; Plasmids ; Retinoblastoma Protein ; Satellite Viruses ; Uterine Cervical Neoplasms*

Numerous studies and clinical trials have demonstrated the efficacy of recombinant adeno-associated virus gene delivery vectors. However, prior to expression, it is necessary to convert the single-stranded DNA genome into double-stranded DNA, which hinders the efficiency of these vectors. We can entirely circumvent this step through the use of self-complementary recombinant adeno-associated virus vector (scrAAV). ScrAAV packages an inverted repeat genome that can fold into double-stranded DNA without the requirement for DNA synthesis or base-pairing between multiple vector genomes. By using scrAAV, we could increase expression efficiency and reduce immune response caused by vectors themselves. Therefore, it is a promising vector for gene therapy. So far, it has been used in the treatment of hepatic diseases, central nervous system diseases, and eye diseases. It has also been used in the modifications of stem cells and as vectors for siRNA/miRNA and ribozymes. In this review, we focused on the preparation, expression and location of scrAAV both in vitro and in vivo. We mainly introduced the recent progress of scrAAV based therapy of Hemophilia B, in order to elucidate the potential and prospects of scrAAV in gene therapy.
Animals ; Base Sequence ; DNA ; genetics ; DNA, Complementary ; genetics ; DNA, Single-Stranded ; genetics ; Dependovirus ; genetics ; metabolism ; Gene Transfer Techniques ; Genetic Therapy ; methods ; trends ; Genetic Vectors ; genetics ; Hemophilia B ; therapy ; Humans ; Molecular Sequence Data

PURPOSE: Wilson disease is an autosomal recessive disorder of copper transport, which is proba bly the most common inherited metabolic disorder in Korea. It is characterized by defective biliary excretion of copper and impairment in the corporation of copper into ceruloplasmin. In Wilson dis ease, synthesis of a defective copper transporting enzyme leads to the accumulation of copper in the liver, brain and kidney. The product of the Wilson disease gene is a copper transporting P- type ATPase(ATP7B). In this study, efforts have been made to identify novel mutations and in vestigate the frequency of the common mutations in Korean patients with Wilson disease. METHODS: This study includes 37 patients from 33 unrelated Korean families with Wilson disease. Genomic DNA from peripheral leukocytes or skin fibroblasts and cDNA from liver tissue were PCR amplified exon by exon, and subsequently analyzed using heteroduplex or SSCP analysis. Speci mens showing mobility shift on those studies were directly sequenced. RESULTS: We identified 12 different mutations in 33 Korean families with Wilson disease; Arg778Leu (R778L), Asn1270Ser(N1270S), Ala874Val(A874V), 2304 del C, 27bp deletion in exon 11, 2461 ins C, Cys656Sop(C656X), Pro768His(P768H), Leu1083Phe(L1083F), Ala1168Ser(A168S), Leu1255Ile(L1255I), and Asp1267Ala(A1267A). Among these, 6 mutations(27bp deletion in exon 11, 2461 ins C, C656X, P768H, A1168S, and L1255I) are novel. The R778L mutation has been known to be highly prevalent in Asian patients. The allele frequency of the R778L in Korean patients with Wilson disease was 37.9%, which was slightly higher than those of Japanese and Taiwanese. Interestingly, the N1270S, originally described in an Italian patient, was the next common mutation in Korean patients withWilson disease with the allele frequency of 12.1%, which was presumed to disrupt ATP hinge domain of the ATP7B protein. The A874V mutation was the third most common mutation with the allele frequency of 9.4%, which was presumed to disrupt Td domain of the ATP7B protein. CONCLUSION: R778L, N1270S, and A874V mutations are three major mutations covering upto nearly 60% of mutated alleles, though Korean patients with Wilson disease are genetically heterogeneous. (J Korean Pediatr Soc 2001;44:569-576)
Adenosine Triphosphate ; Alleles ; Asian Continental Ancestry Group ; Brain ; Ceruloplasmin ; Copper ; DNA ; DNA, Complementary ; Exons ; Fibroblasts ; Gene Frequency ; Hepatolenticular Degeneration* ; Humans* ; Kidney ; Korea ; Leukocytes ; Liver ; Male ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Skin

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.
Biotechnology ; Chimera ; DNA ; DNA, Single-Stranded* ; Genome, Bacterial* ; Retroelements ; Reverse Transcription ; RNA ; RNA-Directed DNA Polymerase

OBJECTIVETo investigate the sequence polymorphism of mtDNA HV1,HV2 overlapping fragments and coding region encompassing position 8430-8673 in Hebei Han population.

METHODSPolymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) combined with sequencing method was used to detect the haplotype distribution of mtDNA in 100 Hebei Han individuals.

RESULTSNinety-one haplotypes were noted in 100 unrelated individuals. The gene diversity is 0.9985 and the random match probability is 0.0115. Compared with the Anderson sequence, 65 sites of different nucleotide sequences were noted, of which 44 sites were previously registered in MITOMAP, 12 sites were not registered and the gene mutations were different from MITOMAP at 9 positions.

CONCLUSIONThe obtained data suggest that these loci are valuable genetic markers for personal identification and thus could be used as basic data for the forensic application of mtDNA in Hebei province.

OBJECTIVES: Mutations in the glucokinase (GCK) gene are considered a possible cause of maturity-onset diabetes of the young. The purpose of this study was to evaluate the contribution of this gene to the development of non insulin dependent diabetes mellitus (NIDDM), gestational diabetes mellitus (GDM) and post-renal transplantation diabetes mellitus (PTDM). METHOD: Identification of GCK mutation was attempted on 39 NIDDM patients, 2 GDM patients and 58 selected renal allograft recipients with PTDM and 45 normal controls. The exons in the GCK gene were examined by polymerase chain reaction (PCR), followed by analysis of single-stranded DNA conformational polymorphism (SSCP). The abnormal bands were also confirmed by DNA sequenc- ing analysis. The exons of affected family members were also investigated for mutations of the GCK gene. RESULTS: Two of the 58 PTDM patients (3.4%) were found to have GCK mutations. One had the mutation on exon 5 and the other on intron 7. One control subject had the mutation on intron 9. The mutation of exon 5 was identified as a substitution of CCT(proline) for CTT (leucine) at codon 164, which has not ever reported before. The family members of the PTDM patient with mutation of exon 5 were analyzed by PCR followed by SSCP, and two of them revealed the same mutation. The abnormal band on the SSCP analysis of exon 7 was identified as the insertion of base C/T at the 39th nucleotide in intron 7. Two family members of this patients also had same band on SSCP. The one mutation of 45 normal controls was CT located at the 8th nucleotide in intron 9, which was a common polymorphism. CONCLUSON: We found GCK mutations in subjects with PTDM and we speculate that these mutations may be one of the contributing cause of PTDM.
Allografts ; Codon ; Diabetes Mellitus ; Diabetes Mellitus, Type 2* ; Diabetes, Gestational ; DNA ; DNA, Single-Stranded ; Exons ; Female ; Glucokinase* ; Humans ; Insulin ; Introns ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Pregnancy

BACKGROUND AND OBJECTIVES: Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel protein, its kinetics and localization are altered in cystic fibrosis. The purpose of this study was to evaluate whether the nasal polyp patients without phenotypic manifestation of cystic fibrosis have any form of CFTR mutations and to characterize the localization of CFTR in the nasal polyp. MATERIALS AND METHODS: The study group consisted of 71 subjects with nasal polyp who underwent an intranasal operation, and 20 normal subjects. Peripheral blood of the study groups were screened for mutation on the exon 3, 4, and 7 of the CFTR gene using single-stranded DNA conformational polymorphism (SSCP). Immunohistochemical staining for CFTR was conducted on the nasal polyps of studied subjects and normal turbinates as the control. RESULTS: While in the nasal polyp group, SSCP screening revealed two cases of mutant band on the exon 3, the normal control group did not show mutant band in all exons screened. CFTR showed the typical apical distribution in the normal turbinate mucosa, whereas in the nasal polyp, regardless of an abnormal band in exon 3, CFTR demonstrated a heterogenous pattern of localization consisting of cytoplasmic labeling, perinuclear staining, and intermingled apical location. CONCLUSION: These results suggest that an altered localization of the CFTR in the nasal polyps, based not only on the CFTR mutation but also on the acquired inflammatory process, may have an important role in the formation of nasal polyps.
Chloride Channels ; Cystic Fibrosis Transmembrane Conductance Regulator* ; Cystic Fibrosis* ; Cytoplasm ; DNA, Single-Stranded ; Exons ; Humans ; Immunohistochemistry ; Kinetics ; Mass Screening ; Mucous Membrane ; Nasal Polyps* ; Polymorphism, Single-Stranded Conformational ; Turbinates

BACKGROUND: Although abnormalities of p53 gene and their relation to clinicopathologic parameters have been identified in some human malignancies, there is little published data on their prevalence and clinical significance in ampullary adenocarcinoma (AAC). The aim of this study is to determine the prevalence of p53 abnormalities in AAC and to evaluate their relation to clinicopathologic features. METHOD:35 formaline-fixed paraffin-embedded tissues of AAC were examined for detection of p53 abnormalities by both single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction- amplified DNA fragments corresponding to exons 5-8 and immunohistochemistry (IHC) using monoclonal antibody to p53 protein (Novocastra, DO7), and the association between the p53 abnormalities and clinicopathologic parameters was analyzed. RESULT: In 22.9% of AAC, p53 gene muation was demonstrated by SSCP analysis, mainly at PCR-amplified exon 8 and exon 7. The p53 protein overexpression by IHC was 48.6% of AAC. Six SSCP and IHC-positive (17.2%) cases and 16 normal (45.7%) cases showed concordant results between the methods, although 13 cases (37.1%) showed discordance, including 11 IHC-positive (31.4%) and 2 SSCP-positive (5.7%) cases. Overall, the prevalence of p53 abnormalities was 54.3%. No significant associations between the p53 abnormalities and clinicopathological parameters such as clinical manifestations, histologic differentiation, and tumor stage were observed. CONCLUSION: The p53 abnormalities detected in 55% of AAC are not associated with prognostic factor, suggesting that abnormal p53 gene may play a role in the development of AAC, but not in its invasiveness.
Adenocarcinoma* ; DNA ; Exons ; Genes, p53* ; Humans ; Immunohistochemistry ; Polymorphism, Single-Stranded Conformational ; Prevalence*

PURPOSE: It is difficult to make the standard ischemic RPE culture model which is still not established. In ischemic model, oxygen and glucose are very important. To improve experimental model, we evaluate the effect of glucose on anoxia-induced human RPE cell damage. METHODS: Human RPE cells were incubated with 95% N2/5% CO2 and standard conditions (95% air/5% CO2) at 37degrees C, and various concentation of glucose were added to a glucose-free medium : no glucose, 0.2 mg/ml, 0.4 mg/ml, -2.0 mg/ml. On a daily basis, morphological and proliferation analysis was conducted using the inverted phase contrast microscope. The extent of cell damage was estimated through cell viability analysis. Cell viability was examined using PMS(phenazinemethosulphate)-MTS(3-(4.5 -dimethyl-thiazol-2-yl)-5-(3-caboxymethoxyphenyl)-2-(4-sulfo phenyl)-2H-tetrazolium, inner salt)assay after 24, 48, 72, and 96 hours. Single-stranded DNAs were detected with monoclonal antibody to prove cell apoptosis in cell damage. RESULTS: At slightly low glucose concentration(0.4 mg/ml), the cell viability of human RPE cells cultured at room air was maintained up to 96 hours, whereas that cultured at 95% N2 decreased after 48 hours. At the anoxic condition. the cell viability of human RPE cells cultured with the low glucose concentration (0.2 mg/ml) decreased after 24 hours, but those done with higher glucose concentrations (more than 0.8 mg/ml) were maintained up to 96 hours. In the cell death caused by the anoxic culture, apoptosis was not suggested by single-stranded DNA detection. CONCLUSIONS: Properly higher glucose concentration on culture medium improve cell viability and reduce the cell damage in human RPE culture in anoxic condition. This result helps to improve experimental ischemic model of RPE cell.
Anoxia ; Apoptosis ; Cell Death ; Cell Survival ; DNA, Single-Stranded ; Glucose* ; Humans* ; Models, Theoretical ; Oxygen ; Retinaldehyde*