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.

China ; DNA, Mitochondrial ; genetics ; Humans ; Polymorphism, Genetic ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational

OBJECTIVETo study the cleavage activity of specific deoxyribozyme to hepatitis C virus in vitro.

METHODSThree deoxyribozymes were designed to cleave at sites 157, 168, 173 in HCV 5'-noncoding region with the active region of 5'-GGCTAGCTACAACGA-3' respectively. Plasmid pCMV/T7-NCRC -Delta Luc was completely linearized with restriction endonuclease Xba I. HCV RNA5'-NCRC was transcribed in vitro from the linearized products and radiolabelled with [alpha-32P] UTP. Under the conditions of 37 degrees C, pH7.5, Mg2+ 10 mmol/L, the three deoxyribozymes were mixed with substrate RNA individually for 120 minutes and then the reactions were terminated. The cleavaged products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. DRz3 was mixed with the substrate RNA at different Mg2+ concentrations. The cleavage efficiency was analyzed with a gel document action analyzing systems.

RESULTSUnder the adopted conditions the three deoxyribozymes efficiently cleaved to the target RNA in vitro and the cleavage activity of DRz3 was increased with the increase of Mg2+ concentration.

CONCLUSIONThe designed deoxyribozymes can cleave 5'-NCR mRNA of HCV efficiently in vitro and it is dose-respondent to Mg2+ concentration.

DNA, Catalytic ; genetics ; DNA, Single-Stranded ; genetics ; Genetic Therapy ; Hepacivirus ; genetics ; Hepatitis C ; therapy ; Humans ; RNA, Messenger ; genetics

OBJECTIVE: To select specific DNA aptamer for determining ketamine by FluMag-SELEX. METHODS: Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. RESULTS: Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine. CONCLUSION FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.
Aptamers, Nucleotide/metabolism* ; DNA ; DNA, Single-Stranded/genetics* ; In Vitro Techniques ; Ketamine/metabolism* ; Oligonucleotides ; SELEX Aptamer Technique/methods*

RecQ5 in mammalian cells has been suggested to suppress inappropriate homologous recombination. However, the specific pathway(s) in which it is involved and the underlining mechanism(s) remain poorly understood. We took advantage of genetic tools in Drosophila to investigate how Drosophila RecQ5 (dRecQ5) functions in vivo in homologous recombination-mediated double strand break (DSB) repair. We generated null alleles of dRecQ5 using the targeted recombination technique. The mutant animals are homozygous viable, but with growth retardation during development. The mutants are sensitive to both exogenous DSB-inducing treatment, such as gamma-irradiation, and endogenously induced double strand breaks (DSBs) by I-Sce I endonuclease. In the absence of dRecQ5, single strand annealing (SSA)-mediated DSB repair is compromised with compensatory increases in either inter-homologous gene conversion, or non-homologous end joining (NHEJ) when inter-chromosomal homologous sequence is unavailable. Loss of function of dRecQ5 also leads to genome instability in loss of heterozygosity (LOH) assays. Together, our data demonstrate that dRecQ5 functions in SSA-mediated DSB repair to achieve its full efficiency and in suppression of LOH in Drosophila.
Animals ; DNA Repair ; genetics ; DNA, Single-Stranded ; genetics ; Drosophila Proteins ; genetics ; metabolism ; Drosophila melanogaster ; genetics ; metabolism ; Loss of Heterozygosity ; genetics ; RecQ Helicases ; genetics ; metabolism

OBJECTIVETo obtain the mutation spectrum of exon 12 of the phenylalanine hydroxylase(PAH) gene.

METHODSThe samples from 127 patients with phenylketonuria(PKU) were studied by polymerase chain reaction(PCR)-single strand conformation polymorphism(SSCP) PCR-denaturing gradient gel electrophoresis(DGGE) and direct DNA sequencing.

RESULTSFour heterozygous mutations including R413P S411X R408W R408Q in 10 of 127 patients were identified by DNA sequencing. The frequencies of R413P S411X R408W R408Q mutation were 2.76%, 0.39%, 0.39% and 0.39%, respectively. The S411X mutation in Chinese was first reported. Only 2 cases with R413P mutation were detected by SSCP. DGGE analysis showed that 10 cases displayed 3 kinds of abnormal electrophoretic bands. There were no obvious differences in the frequency of R413P mutation between northern and southern Chinese with PKU, and between classical PKU and hyperphenylalaninemia.

CONCLUSIONDGGE is more sensitive than SSCP in screening the mutations of exon 12 of the PAH gene. DGGE combined with DNA sequencing can be used to define all the mutations of exon 12 of the PAH gene. These results indicate the similarity in the frequency of R413P mutation between northern and southern patients.

Electrophoresis, Polyacrylamide Gel ; Exons ; Humans ; Mutation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; genetics ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA

OBJECTIVETo explore new mutation in phenylalanine hydroxylase (PAH) gene.

METHODSThe PAH genes from 40 phenylketonuria (PKU) patients and 30 normal controls were screened by PCR-single strand conformation polymorphism (SSCP) and further sequencing.

RESULTSEleven mutations and 3 polymorphisms in PAH gene were found. No abnormalities in the PAH gene from 30 controls were detected.

CONCLUSIONM276K, M276R, 280insT, IVS10nt+32T-->A, IVS4nt+47C-->T were demonstrated as novel mutations in comparison with the PAH mutation database. One mission mutation (H290R) was first documented in Chinese PKU gene.

DNA Mutational Analysis ; Female ; Humans ; Infant ; Male ; Mutation ; Phenylalanine Hydroxylase ; genetics ; Phenylketonurias ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single-Stranded Conformational

OBJECTIVE: To study the application of PCR-SSCP in forensic mtDNA typing. METHODS: Primers flanking the mtDNA HV-I and HV-II regions were designed. By PCR-SSCP techniques, 70 family trios and 140 unrelated Wuhan Han individuals were investigated and analyzed. RESULTS: In 70 family trios, the SSCP profiles in region HV-I and HV-II of children were not same to that of their fathers in 98.57% and 97.13% respectively but were identical with their mothers. In 140 unrelated Wuhan Han individuals, 21 haplotypes were found in HVI, GD = 0.9556; 16 haplotypes were found in HVII, GD = 0.9356. CONCLUSION PCR-SSCP technique may be useful in forensic mtDNA typing, especially for screening the suspects.
DNA Fingerprinting/methods* ; DNA Primers ; DNA, Mitochondrial/genetics* ; Forensic Genetics/methods* ; Haplotypes ; Humans ; Pedigree ; Polymerase Chain Reaction/methods* ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA

We have assessed the relationships between immune trait (antibody titers of Sheep red blood cell, SRBC; Avian influenza, AI; Newcastle disease, ND) and varieties of MHC B-LBHII Gene in local chicken breeds (Wenshang Barred chicken, LH; Laiwu Black chicken, LWH; and Jining Bairi chicken, BR). We selected 300 chickens randomly from the three indigenous chicken populations. The variations of MHC B-L BII gene were detected by directly DNA sequencing and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). The results indicated that there were about 19-22 nucleotide mutations in the three local breeds, which could affect 16-18 amino acid variations. Another results indicated that there was significantly relationship between seven to eight SNPs of the MHC B-LBII region and some immune traits (P < 0.05 or P < 0.01). Both locus G97A and locus T138A were found in the three species, which were significantly related to the antibodies of SRBC, ND and AI antibody titers (P < 0.05). Among them, the locus G97A was significantly associated with ND antibody titers (P < 0.05) in BR chicken, with SRBC antibody titers (P < 0.05) in LWH chicken, and with H9 antibody titers (P < 0.05) in LH chicken. Furthermore, locus T138A was significantly associated with H9 antibody titers in BR and LH chickens (P < 0.05). All those results suggest relationships among the different varieties of MHC B-LBII and immune traits in the three local breeds.
Animals ; Base Sequence ; Breeding ; Chickens ; genetics ; immunology ; Major Histocompatibility Complex ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Polymorphism, Single-Stranded Conformational ; Sequence Analysis, DNA

OBJECTIVETo investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.

METHODS10-23 DNA enzymes named DrzBC-7, DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A1816UG were designed and synthesized. HBV C gene mRNA was obtained by in vitro transcription method. Cleavage activities were observed in vitro. The influence of MgCl2 concentration on RNA cleaving activity was examined with DrzBC-9. Values of kinetic parameters including Km, Kcat and Kcat/Km were calculated accordingly.

RESULTTargeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro. Under the certain cleavage conditions, DrzBC-7, DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro. Cleavage products of 109 nt and 191 nt were obtained. No cleavage occurred without MgCl2. The most efficient cleavage was obtained at 150 mmol x L(-1) MgCl2. The efficiency of cleavage did not increase when the MgCl2 concentration was more than 200 mmol x L(-1). The kinetic parameters, Km, Kcat and Kcat/Km for DrzBC-9 were 1.4x10(-9) mol x L(-1), 1.6 min(-1) and 1.1x10(9) mol x L(-1) x min(-1), respectively.

CONCLUSION10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.

DNA, Catalytic ; metabolism ; DNA, Single-Stranded ; metabolism ; Hepatitis B virus ; enzymology ; genetics ; Open Reading Frames ; RNA, Messenger ; metabolism ; RNA, Viral ; genetics ; metabolism ; Transcription, Genetic

OBJECTIVETo develop a simple, cheap and efficient restriction endonucleases fingerprinting-single strand conformation polymorphism(REF-SSCP) method applied to screen for mutations in long segments.

METHODSThe genomic DNA of Cx26 gene segment of the patients with deafness was amplified. The amplification products were screened with SSCP and REF-SSCP technique and DNA sequencing to evaluate and compare the effect on detection of mutations in long segments.

RESULTSNo different band was found in 724 bp segment in SSCP examination. Three kinds of different bands were discovered in REF-SSCP examination and the 79 G -->A mutation detected by DNA sequencing were accorded with the REF-SSCP bands entirely. The rate of detection was 100%.

CONCLUSIONThe present REF-SSCP method is applicable to screen mutations in long segment DNA of mass specimens.

Connexin 26 ; Connexins ; genetics ; DNA Fingerprinting ; methods ; DNA Restriction Enzymes ; Deafness ; genetics ; Genetic Testing ; methods ; Humans ; Mutation ; Polymorphism, Single-Stranded Conformational ; Sensitivity and Specificity