Single nucleotide polymorphisms (SNP) is an important molecular marker in traditional Chinese medicine research, and it is widely used in TCM authentication. The present study created a new genotyping method by combining restriction endonuclease digesting with melting curve analysis, which is a stable, rapid and easy doing SNP genotyping method. The new method analyzed SNP genotyping of two chloroplast SNP which was located in or out of the endonuclease recognition site, the results showed that when attaching a 14 bp GC-clamp (cggcgggagggcgg) to 5' end of the primer and selecting suited endonuclease to digest the amplification products, the melting curve of Lonicera japonica and Atractylodes macrocephala were all of double peaks and the adulterants Shan-yin-hua and A. lancea were of single peaks. The results indicated that the method had good stability and reproducibility for identifying authentic medicines from its adulterants. It is a potential SNP genotyping method and named restriction endonuclease digest - melting curve analysis.
Atractylodes ; classification ; genetics ; DNA Restriction Enzymes ; metabolism ; DNA, Plant ; genetics ; Drug Contamination ; Genotype ; Lonicera ; classification ; genetics ; Plants, Medicinal ; classification ; genetics ; Polymorphism, Single Nucleotide

In our screening for photosensitizers from natural resources, four alkaloids were isolated from Glycosmis pentaphylla by various chromatography techniques. Their structures were identified as glycoborinine (1), glybomine B (2), carbalexin A (3) and N-p-coumaroyltyramine (4) by spectral analysis. Their photoactivated antimicrobial activities were evaluated by thin-layer chromatography (TLC) agar overlay assay against Staphylococcus aureus and Bacillus subtilis. It was found that compounds 1 and 4 showed photo-activated antimicrobial activities. Meantime, photo-activated DNA binding activities of these compounds were also assessed by using a specially prepared 1.8 kb DNA fragment and restriction enzymes. Under UVA irradiation, compound 1 showed moderate inhibition on Nde I, Xba I, Nco I and Bcl I which have either 5'-TpA or 5'-ApT and trace or no inhibition on other restriction enzymes. It showed a similar inhibition pattern with the reference 8-methoxypsoralen. However, compounds 2-4 showed no inhibition against any of the restriction enzymes.
Anti-Bacterial Agents ; chemistry ; isolation & purification ; pharmacology ; Bacillus subtilis ; drug effects ; Carbazoles ; chemistry ; isolation & purification ; pharmacology ; Chromatography, Thin Layer ; Coumaric Acids ; chemistry ; isolation & purification ; pharmacology ; DNA Fragmentation ; DNA Restriction Enzymes ; metabolism ; Light ; Molecular Structure ; Photosensitizing Agents ; pharmacology ; Plants, Medicinal ; chemistry ; Protein Binding ; Rutaceae ; chemistry ; Staphylococcus aureus ; drug effects ; Ultraviolet Rays

Carcinoma, Hepatocellular ; genetics ; metabolism ; DNA Methylation ; DNA Restriction Enzymes ; metabolism ; DNA, Neoplasm ; genetics ; Glutathione S-Transferase pi ; genetics ; metabolism ; Humans ; Liver Neoplasms ; genetics ; metabolism ; Polymerase Chain Reaction ; methods

Recently, emerging waterborne protozoa, such as microsporidia, Cyclospora, and Cryptosporidium, have become a challenge to human health worldwide. Rapid, simple, and economical detection methods for these major waterborne protozoa in environmental and clinical samples are necessary to control infection and improve public health. In the present study, we developed a multiplex PCR test that is able to detect all these 3 major waterborne protozoa at the same time. Detection limits of the multiplex PCR method ranged from 101 to 102 oocysts or spores. The primers for microsporidia or Cryptosporidium used in this study can detect both Enterocytozoon bieneusi and Encephalitozoon intestinalis, or both Cryptosporidium hominis and Cryptosporidium parvum, respectively. Restriction enzyme digestion of PCR products with BsaBI or BsiEI makes it possible to distinguish the 2 species of microsporidia or Cryptosporidium, respectively. This simple, rapid, and cost-effective multiplex PCR method will be useful for detecting outbreaks or sporadic cases of waterborne protozoa infections.
Cryptosporidium/*isolation & purification ; Cyclospora/*isolation & purification ; DNA Primers/genetics ; DNA Restriction Enzymes/metabolism ; DNA, Protozoan/genetics/metabolism ; Humans ; Microsporidia/*isolation & purification ; Parasitology/*methods ; Polymerase Chain Reaction/*methods ; Polymorphism, Restriction Fragment Length ; Sensitivity and Specificity ; Water/*parasitology

OBJECTIVETo construct an expression vector of the gene encoding rat interferon-gamma-inducible protein (IP-10) and identify its expression in NIH 3T3 cells.

METHODSIP-10 gene was amplified by polymerase chain reaction (PCR) and inserted into the eukaryotic expression vector pcDNA3.1(+). After identification by PCR, restriction endonuclease digestion and sequence analysis, the recombinant expression vector pcDNA3.1(+)-IP-10 was transfected into NIH 3T3 cells via liposome. Immunofluorescence method was used to confirm the expression of pcDNA3.1(+)-IP-10 in the transfected NIH 3T3 cells. The expression of IP-10 protein in the supernatant of the transfected cells was examined by Western blotting.

RESULTSPCR, restriction endonuclease digestion and sequence analyses confirmed successful construction of the recombinant vector pcDNA3.1(+)-IP-10. Immunofluorescence assay identified the expression of pcDNA3.1(+)-IP-10 in NIH 3T3 cells, and the expression of IP-10 protein was detected by Western blotting in the supernatant of the transfected cells.

CONCLUSIONA eukaryotic expression vector pcDNA3.1(+)-IP-10 has been successfully constructed, which provides the basis for investigating the therapeutic effect of IP-10 on Th1 type autoimmune disease.

Animals ; Chemokine CXCL10 ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; Fluorescent Antibody Technique ; Gene Expression ; Genetic Vectors ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; genetics ; Rats ; Transfection ; methods

In order to prevent phage contamination in amino acid fermentation, we introduced the restriction and modification system cglI gene complex into Corynebacterium crenatum and studied their phage-resistance. The cglI gene complex was amplified from Corynebacterium glutamicum by PCR and constructed into pJL23 vector. The recombinant strains were obtained by transformation of the recombinant plasmid pJL23-cglI into C. crenatum. Results showed that the recombinant strains possessed strong phage-resistance activity and broad phage-resistance spectrum, demonstrating the feasibility of using cglI gene complex for construction of phage-resistance recombinant C. crenatum strains and presenting a powerful way to solve the problem of phage contamination in amino acid fermentation industry.
Amino Acids ; biosynthesis ; Bacterial Proteins ; genetics ; Bacteriophages ; growth & development ; Corynebacterium ; genetics ; virology ; Corynebacterium glutamicum ; genetics ; metabolism ; DNA Restriction-Modification Enzymes ; genetics ; Fermentation ; Galectins ; genetics ; Recombination, Genetic ; Transformation, Bacterial

OBJECTIVETo construct a recombinant adenovirus vector bearing dual-survivin short hairpin RNA (shRNA).

METHODSDual-survivin shRNA was designed and synthesized respectively, both inserted into adenovirus DNA. The recombinant adenovirus vector was confirmed via both sequencing and restriction digestion analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenoviruses.

RESULTSThe recombinant adenovirus vector was constructed and the target sequence was obtained.

CONCLUSIONThe construction of the recombinant adenovirus vector provides a basis for the research of potential gene therapy for prostate cancer.

Adenoviridae ; genetics ; Cell Line ; DNA Restriction Enzymes ; metabolism ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; genetics ; Polymerase Chain Reaction ; RNA, Small Interfering ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo construct the recombinant plasmid containing human microdystrophin cDNA, and study the microdystrophin expression in vivo and in vitro.

METHODSMicrodystrophin cDNA was obtained from recombinant plasmid pBSK-MICRO digested with restrictive endonuclease Not I, the product was inserted into plasmid pVAX1, resulting in pAMICDYS. And then 3T3 cells were transfected with pAMICDYS. Forty-eight hours after transfection, the expression of the microdystrophin was detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Finally, TA muscles of mdx mice were injected with the recombinant plasmid pAMICDYS through i.m. and the pathological change of TA was evaluated by histology, and the expression of microdystrophin in mdx TA was detected by immunohistochemical analysis.

RESULTSThe recombinant plasmid containing human microdystrophin cDNA was constructed successfully. The recombinant plasmid was proved to be able to express microdystrophin protein both in vivo and in vitro. Moreover, treatment of the TA of mdx mice with the recombinant plasmid could decrease the number of centrally nucleated myofibers.

CONCLUSIONRecombinant plasmid containing the microdystrophin gene was constructed successfully, and it could express microdystrophin protein both in vivo and in vitro. It provides basis for further study on microdystrophin as a target gene to treat Duchenne muscular dystrophy (DMD) by electrotransfer, i.v, arterial injection and combining with other exogenous gene to enhance microdystrophin expression.

Animals ; Cloning, Molecular ; DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; DNA, Recombinant ; genetics ; metabolism ; Dystrophin ; genetics ; Gene Expression ; Genetic Engineering ; Genetic Therapy ; Genetic Vectors ; metabolism ; Humans ; Immunohistochemistry ; Mice ; Muscular Dystrophy, Duchenne ; genetics ; metabolism ; therapy ; NIH 3T3 Cells ; Plasmids ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

OBJECTIVETo detect the most prevalent mutations, R778L and P992L of ATP8B gene, in Chinese Wilson disease(WD) patients by high resolution melting (HRM) analysis after polymerase chain reaction (PCR).

METHODSGenomic DNA was extracted from peripheral blood samples obtained from 30 cases of WD by the standard phenol/chloroform method. DNA fragments encompassing ATP7B exons 8 and 13 were produced by PCR amplification. The amplicons containing the R778L or P992L mutations were then generated by nested PCR. The nested PCR products were subjected to HRM analysis using the HR-1 instrument. Mutations detected in HRM analysis were verified by restriction analysis using restriction enzyme (MspI or AluI or AfaI) or DNA sequencing.

RESULTSHRM analysis of the fragments encompassing ATP7B exon 8 showed four curve patterns. Subsequent restriction analysis and DNA sequencing proved that the four different curves represent four different genotypes: the wild type, the R778L/R778L homozygote, the R778L heterozygote, and the R778L/752.33delG compound heterozygote. Three HRM curve patterns were observed for the fragments encompassing ATP7B exon 13, representing the wild type, the P992L heterozygote, and the P992L/S975Y compound heterozygote. In our studied samples, allele frequencies of the R778L, P992L and S975Y mutations were 25%, 15% and 1.67%, respectively.

CONCLUSIONHRM analysis is a simple, accurate and sensitive approach for rapid detection of the ATP7B mutations and could be used as an optimized method for genetic testing in WD.

Adenosine Triphosphatases ; genetics ; Base Sequence ; Cation Transport Proteins ; genetics ; Copper-transporting ATPases ; DNA ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; DNA Restriction Enzymes ; metabolism ; Exons ; genetics ; Freezing ; Gene Frequency ; Genotype ; Hepatolenticular Degeneration ; genetics ; Humans ; Nucleic Acid Denaturation ; Polymerase Chain Reaction ; Time Factors

OBJECTIVETo analyze two alleles (4qA and 4qB) distal to D4Z4 of the 4q subtelomere in Chinese population, and to elucidate the interrelationship between these variants of 4qter and facioscapulohumeral muscular dystrophy (FSHD).

METHODSEighty unrelated healthy individuals from a random Chinese Han population were investigated. The genomic DNA was extracted from peripheral blood lymphocytes according to the specific procedure designed to minimize DNA shearing, then digested with EcoRI, HindIII or double digested with EcoRI and BlnI. The cleaved DNA was separated by pulsed field gel electrophoresis (PFGE) and Southern blotting with the probes p13E-11, 4qA and 4qB. The sizes of 4q35 EcoRI/4qA and EcoRI/4qB arrays were obtained by "curve fitting", and the frequencies of alleles and genotypes were calculated. Data were analyzed using a commercially available statistical package (Version 13.0 SPSS).

RESULTSIn normal individuals, frequencies of 4qA and 4qB alleles (46.9% and 53.1%) were observed of no significant difference (chi(2) = 1.250, P>0.05). The frequency of 4qA/4qB heterozygote was much higher than that of homozygote (P<0.05). The means of EcoRI/4qA and EcoRI/ 4qB arrays (115.8+/-11.9 kb and 98.3+/-8.6 kb) were of significant difference (t=23.04, P<0.001). 8.8% (7/80) of the individuals displayed a translocation repeat array configuration. 4qB-type EcoRI arrays smaller than 35 kb were found in two individuals.

CONCLUSIONThe structural polymorphism of 4qA/4qB alleles within 4q35 and 10q26 is confirmed using PFGE in normal Chinese Han population. Although both alleles are almost equally common, shorten 4qB-type EcoRI fragment is not pathogenic. The frequency of 4qA/4qB heterozygote is significantly higher than that of homozygote.

Adult ; Alleles ; Asian Continental Ancestry Group ; genetics ; China ; ethnology ; Chromosomes, Human, Pair 4 ; genetics ; DNA ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; Escherichia coli ; enzymology ; Ethnic Groups ; genetics ; Female ; Humans ; Male ; Middle Aged ; Muscular Dystrophy, Facioscapulohumeral ; genetics ; Sex Distribution ; Telomere ; genetics