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

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

29 isoniazid (INH) resistant isolated strains and INH sensitive reference strain (H37Rv) of Mycobacterium tuberculosis were analysed by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and NciI restriction mapping for the detection of mutations in katG gene and inhA gene. The katG gene was divided into 3 parts (Akat, Bkat, Ckat; each part is about 800 bp) and amplified, inhA gene was amplified as a whole. Each of the amplified 800 bp DNA was digested into small fragments of less than 400 bp with restriction enzymes for the direct PCR-SSCP analysis. Firstly, 10 strains were analysed. All the 10 isolates showed clearly distinct SSCP patterns in Bkat from that of the reference strain, but only two isolates showed distinct SSCP patterns in Akat, and no isolated strain showed any distinct SSCP patterns in Ckat. 10 isolates also showed distinct SSCP patterns in inhA. NciI restriction mapping of Bkat showed mutation in codon 463 in 7 strains among 10 isolated strains. With these results an early detection strategy for the INH resistant M. tuberculosis was applied to the rest of 19 isolated INH resistant strains. Firstly, isolates were screened by Ncsl mapping in Bkat, and 13 strains showed mutations in codon 463. Secondly, the rest of 6 INH resistant isolates were analysed by PCR-SSCP with restriction enzyme digestion (PCR-SSCP-RE) in Bkat, and all the strains showed distinct SSCP patterns from that of the INH sensitive reference strain. This proved our strategy as effective and economic and time saving method in early detection of INH resistant M. tuberculosis.
Codon ; Diagnosis* ; Digestion ; DNA ; Isoniazid* ; Korea* ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymorphism, Single-Stranded Conformational ; Restriction Mapping ; Tuberculosis

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*

PURPOSE: The purpose of this paper is to identify the forkhead transcription factor gene (FOXL2) mutations in Korean patients with blepharophimosis-ptosis-epicanthus inversus syndrome (BPES). METHODS: We have analyzed the mutations of FOXL2 gene in genomic DNAs extracted from 16 BPES patients and their families by PCR, PCR-SSCP, and sequencing. RESULTS: No deletion in exon 1 to 3 of the FOXL2 gene was observed by PCR. The PCR products were subjected to SSCP analysis and 9 patients showed SSCP shifts. The PCR products showing SSCP shifts were subcloned into plasmid vectors and sequenced to confirm the FOXL2 mutation. In total, 7 mutations (1 nonsense mutation, 1 deletion, and 5 duplications) in exon 2 were identified. CONCLUSIONS: The FOXL2 gene mutations were identified in the Korean BPES patients. Some of the mutations were previously reported and some were new mutations. This study will contribute to the molecular analysis and clinical counseling of BPES patients.
Codon, Nonsense ; Counseling ; DNA ; Exons ; Humans ; Plasmids ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Transcription Factors

BACKGROUND: The 463 codon mutation of katG gene has been reported as an useful marker for the detection of isoniazid(INH) resistant strains of M. tuberculosis. This study aimed to elucidate relationship between 463 mutation in katG gene and INH resistance in M. tuberculosis. METHOD: DNA was extracted from 28 INH susceptible strains(MIC > or = 0.2microg/ml on the Lowenstein Jensen media) and used for amplification of 189bp fragment containing 463 codon by PCR. Amplified fragments were digested by restriction enzyme Msp I, analyzed by single strand conformation polymorphism(SSCP) in the MDE gel and sequenced to prove mutation. RESULT: Only 7(25%) out of 28 were digestible by restriction enzyme Msp I. The SSCP pattern of 21 strains were distinctly different from that of M. tuberculosis H37Rv. Msp I undigestible PCR fragment was substituted at 463 codon from Arg(CGG) to Leu(CTG). CONCLUSION: This finding clearly indicate no relationship between 463 codon mutation of the katG gene and INH resistance.
Codon ; DNA ; Isoniazid* ; Mycobacterium tuberculosis* ; Mycobacterium* ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational ; Tuberculosis