Cloning of large genomic sequences is an enabling technology in synthetic biology. To obtain large gene fragments, traditional cloning methods are faced with various defects, for instance, random library cloning relies always on high-throughput screening. It is difficult to get gene fragments more than 10 kb by PCR amplification. Assembly of small fragments is labor intensive with high mutation rates. It is difficult to find suitable cleavage sites on the fragment ends by restriction endonuclease. Recently genome-wide editing creates a new high-performance large fragments cloning methods. For example, CRISPR/cas9 system can identify and cut 20 bp nucleic acid sequences recognition sites used to obtain any desired gene fragments; if combined with Gibson or transformation associated recombination (TAR) assembly technology, these methods can efficiently clone large fragments. This article introduces large fragments cloning technology by classification, then proposes the choice criteria of methods for cloning gene fragments of different sizes.
CRISPR-Cas Systems ; Cloning, Molecular ; DNA Restriction Enzymes ; Multigene Family ; Polymerase Chain Reaction ; Synthetic Biology

PURPOSE: Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori-specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target. MATERIALS AND METHODS: The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-beta-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC. RESULTS: The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein. CONCLUSION: Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.
Chromatography, Liquid ; Clone Cells* ; Cloning, Organism* ; Digestion ; DNA ; DNA Restriction Enzymes ; Duodenum ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli* ; Escherichia* ; Helicobacter pylori* ; Helicobacter* ; Humans ; Lipoproteins ; Plasmids ; Polymerase Chain Reaction ; Sodium Dodecyl Sulfate ; Stomach ; Vaccination*

OBJECTIVE: To investigate the interaction between polymorphism of Toll-like receptor 4 (TLR4) gene G11367C in 3' untranslated region (UTR) and inhibitor of nuclear factor kappaB (IκB)-α 
Hae III in acute pancreatitis (AP) and the degree of severity.
 METHODS: A total of 450 patients with confirmed AP (AP group), who came from the First Affiliated Hospital of Xinxiang Medical College from May 2013 to June 2015, were divided into a mild AP subgroup (MAP subgroup), a moderately severe AP (MSAP subgroup), and a severe acute AP (SAP subgroup) (n=150 in each group). One hundred fifty healthy persons were served as a control group. There was no significant difference in age, gender, ethnicity and birthplace among all groups. The genetic polymorphisms of TLR4 gene G11367C in 3' untranslated region and IκB-α Hae III were analyzed by polymerase chain reaction (PCR). Eligible participants were personally interviewed by a questionnaire. Unconditional logistic regression model and single factor analysis were performed to calculate the adjusted odds ratios (OR) and 95% confidence intervals (95% CI) of G11367C and IκB-α Hae III polymorphisms, respectively. The interaction of nucleotide polymorphisms was analyzed.
 RESULTS: The frequencies of G11367C (GC), IκB-α Hae III (AG) and IκB-α Hae III (GG) were 69.56%, 33.78% and 36.22% in the AP group; 49.33%, 24.67% and 26.00% in the MAP subgroup; 70.67%, 34.67% and 36.67% in the MSAP subgroup; 88.67%, 42.00% and 46.00% in the SAP subgroup and 26.67%, 14.00% and 14.67% in the control group, respectively. There was significant difference in the frequencies betweenc the AP group and the control group, or among each AP subgroup (all P<0.01). The risk of AP was significantly increased in the subjects with G11367C (GC) genotype (ORAP=6.2828, ORMAP=2.6776, ORMSAP=6.6250, ORSAP=21.5147), which was also increased in those with IκB-α Hae III (AG) genotype (ORAP=5.7369, ORMAP=2.5277, ORMSAP=6.1824, ORSAP=17.8572) and in those with IκB-α Hae III (GG) genotype (ORAP=5.8724, ORMAP=2.5902, ORMSAP=6.4027, ORSAP=18.9022). The combined analysis of the polymorphisms showed that the percentage of G11367C (GC)/ IκB-α Hae III (GG) in the AP group, the MAP subgroup, the MSAP subgroup, the SAP subgroup and the control groups was 26.44%, 12.67%, 26.00%, 40.67% and 4.00%, respectively, with significant difference in the frequency among all groups (all P<0.01). The people who carried with Pro12Ala (AA)/Pro198Leu (LL) had a high risk of AP (ORAP=30.1314, ORMAP=6.7612, ORMSAP=39.5000, ORSAP=401.5833), and the statistical analysis suggested a positive interaction between Pro12Ala (AA) and Pro198Leu (LL) in increasing the risk of AP (All γ>1). Similarly, there were also positive interactions in the pathogenesis of AP between G11367C (GC) and IκB-α Hae III (AG) (All γ>1). 
 CONCLUSION These carriers of G11367C(GC), IκB-α Hae III(AG) and IκB-α Hae III (GG) genotypes may have a high risk of AP occurency, and there are significant interactions between genetic polymorphisms of G11367C and IκB-α Hae III, which increaes the risk of the occurrence and development of AP.
3' Untranslated Regions ; Acute Disease ; Deoxyribonucleases, Type II Site-Specific ; Ethnic Groups ; Genetic Predisposition to Disease ; Genotype ; Humans ; I-kappa B Kinase ; Logistic Models ; NF-KappaB Inhibitor alpha ; Odds Ratio ; Pancreatitis ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Promoter Regions, Genetic ; Toll-Like Receptor 4

In developing countries threat of cholera is a significant health concern whenever water purification and sewage disposal systems are inadequate. Vibrio cholerae is one of the responsible bacteria involved in cholera disease. The complete genome sequence of V. cholerae deciphers the presence of various genes and hypothetical proteins whose function are not yet understood. Hence analyzing and annotating the structure and function of hypothetical proteins is important for understanding the V. cholerae. V. cholerae O139 is the most common and pathogenic bacterial strain among various V. cholerae strains. In this study sequence of six hypothetical proteins of V. cholerae O139 has been annotated from NCBI. Various computational tools and databases have been used to determine domain family, protein-protein interaction, solubility of protein, ligand binding sites etc. The three dimensional structure of two proteins were modeled and their ligand binding sites were identified. We have found domains and families of only one protein. The analysis revealed that these proteins might have antibiotic resistance activity, DNA breaking-rejoining activity, integrase enzyme activity, restriction endonuclease, etc. Structural prediction of these proteins and detection of binding sites from this study would indicate a potential target aiding docking studies for therapeutic designing against cholera.
Bacteria ; Binding Sites ; Cholera ; Computer Simulation* ; Developing Countries ; DNA ; DNA Restriction Enzymes ; Drug Discovery ; Drug Resistance, Microbial ; Genome ; Humans ; Integrases ; Sewage ; Solubility ; Vibrio cholerae ; Vibrio cholerae O139* ; Water Purification

OBJECTIVETo assess the association of vitamin D receptor gene (VDR) Apa I, Bsm I genotypes and allele frequencies and mild cognitive impairment (MCI) among elderly ethnic Uygurs from Xinjiang, China.

METHODSThe polymorphisms of the VDR genotypes (Apa I and Bsm I) were analyzed by the SNaPshot method in 124 MCI patients and 124 controls.

RESULTSFactors which can increase the risk for MCI have included the A allele of the Apa I polymorphism [OR=1.62, 95%CI(1.13-2.31)] and the AA genotype [OR=3.49, 95% CI(1.57-7.74)], the T allele of the Bsm I polymorphism [OR=1.94, 95%CI(1.24-3.05)], higher triglyceride and systolic blood pressure levels.

CONCLUSIONPolymorphisms of the VDR gene including the A allele and AA genotype of Apa I, and the T allele of Bsm I are probably associated with MCI among elderly ethnic Uygurs, and so are higher levels of triglyceride and systolic blood pressure.

Aged ; Alleles ; Asian Continental Ancestry Group ; genetics ; Binding Sites ; genetics ; Blood Pressure ; China ; Cognitive Dysfunction ; ethnology ; genetics ; psychology ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Diagnostic and Statistical Manual of Mental Disorders ; Ethnic Groups ; genetics ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; ethnology ; genetics ; Genotype ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Polymorphism, Single Nucleotide ; Receptors, Calcitriol ; genetics ; Triglycerides ; blood

To reveal the effect of rotation cropping and bacterial manure on the growth of Chrysanthemum morifolium and screen the beneficial endophytic, the diversity of endophytic and dominant genera of different treatment groups were analyzed. Four different treatments were continuous cropping, rotation, self-made organic fertilizer and commercially available fertilizer, respectively. Endophytic bacterial diversity and dominant genera in different organs were examined using Terminal Restriction Fragment Length Polymorphism (T-RFLP). The results showed that enzyme Hae III was more appropriate than enzyme Hinfl because the number of TRFs digested by enzyme Hae III was more than that of enzyme Hinfl. In comparison of diversity, the endophytic bacterial communities' diversity index in group of cropping rotation and fertilizer was higher than that of continuous cropping which indicated that the addition of exogenous microorganism in soil could increase the diversity of plant endophyte. 18 dominant species were selected, including 3 kinds of Firmicutes, 4 kinds of Actinomycetes and 11 kinds of Proteobacteria. The results of dominant species comparison showed that the number of dominant species in continuous cropping of Ch. morifolium was significantly less than that of the rotation group. Some dominant bacteria in rotation group and fertilizer group such as Arthrobacter, Streptomyces, Streptomyces, Flavobacterium and Mycobacterium were not found in the continuous cropping of Ch. mortfolium group. Dominant species of fertilizer treatment group was similar with the rotation group, and the continuous cropping group's dominant species was more abundant. It indicates that these bacteria may be able to mitigate hindrance in continuous cropping, especially the Flavobacterium which can decompose the pathogenic fungi is worthy of further attention. Compared with leaves, there are more dominant species in roots and stems. The diversity of edophytic bacterial communities in continuous cropping of Ch. morifolium stays below than that in the rotation of Ch. morifolium, and fertilizer treatment can increase the diversity of continuous cropping so that it could mitigate hindrance in continuous cropping.
Actinobacteria ; physiology ; Agriculture ; Biodiversity ; Chrysanthemum ; growth & development ; microbiology ; Deoxyribonucleases, Type II Site-Specific ; Endophytes ; Fertilizers ; Gram-Positive Bacteria ; physiology ; Phylogeny ; Plant Leaves ; Plant Roots ; microbiology ; Polymorphism, Restriction Fragment Length ; Proteobacteria ; physiology ; RNA, Ribosomal, 16S ; chemistry ; genetics ; Soil ; Soil Microbiology

Gene cloning is one of the most important and widely used technologies in molecular biology research. Generally, DNA fragment is cut with restriction enzyme, and then the product is ligated to a linearized vector with complementary sticky end or blunt end by DNA-ligase. This traditional DNA cloning method requires compatible enzyme recognition sites existing in both PCR fragment and targeted vector. Several ligase-free methods have been established to avoid the using of restriction enzyme. However, those methods are time-consuming, labor-intensive and expensive. To overcome these shortcomings, we developed an Exonuclease III based DNA cloning method that takes only 30 minutes with high cloning efficiency and significant economic advantage. Therefore, this method is suitable for large-scale gene cloning.
Cloning, Molecular ; methods ; DNA ; chemistry ; DNA Restriction Enzymes ; Exodeoxyribonucleases ; chemistry ; Genetic Vectors ; Polymerase Chain Reaction

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

OBJECTIVETo investigate the single nucleotide polymorphisms (SNPs) at interleukin 6 (IL-6)-174 and TNF-β NcoI in Chinese Han children in Guangzhou, China and to provide basic information for study on the association between IL-6-174 and TNF-β NcoI polymorphisms and systemic inflammatory response syndrome (SIRS).

METHODSAllele-specific polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism were used to determine the SNPs at IL-6-174 and TNF-β NcoI in 481 children selected from the Han population in Guangzhou in 2012. Genotype analysis and comparison with other populations were made with reference to relevant literature.

RESULTSChinese Han children in Guangzhou had only GG genotype at IL-6-174, and the SNP at this locus was rare or not seen in the Han population in Guangzhou. At TNF-β NcoI, the frequencies of TNF-β 1*1, TNF-β 1*2, and TNF-β 2*2 genotypes were 24.7%, 49.7%, and 25.6%, respectively. The sample distribution was in accordance with Hardy-Weinberg equilibrium. The TNF-β 1 allele frequency was significantly higher in Guangzhou Han population than in European and American white population (P<0.05).

CONCLUSIONSTNF-β NcoI SNP is prevalent in the Han population in Guangzhou, and the distribution of alleles is significantly different from that in the white population. The sample from an Hardy-Weinberg equilibrium population can be further used for study on the association between TNF-β NcoI SNP and SIRS in Chinese Han children in Guangzhou. IL-6-174 SNP is rare or not seen in the Han population in Guangzhou, so SNP at this locus cannot be selected for disease association analysis.

Asian Continental Ancestry Group ; genetics ; Child, Preschool ; China ; ethnology ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Female ; Gene Frequency ; Humans ; Interleukin-6 ; genetics ; Lymphotoxin-alpha ; genetics ; Male ; Polymorphism, Single Nucleotide ; Systemic Inflammatory Response Syndrome ; genetics

DraIII is a type IIP restriction endonucleases (REases) that recognizes and creates a double strand break within the gapped palindromic sequence CAC↑NNN↓GTG of double-stranded DNA (↑ indicates nicking on the bottom strand; ↓ indicates nicking on the top strand). However, wild type DraIII shows significant star activity. In this study, it was found that the prominent star site is CAT↑GTT↓GTG, consisting of a star 5' half (CAT) and a canonical 3' half (GTG). DraIII nicks the 3' canonical half site at a faster rate than the 5' star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the DraIII protein was solved. It indicated, as supported by mutagenesis, that DraIII possesses a ββα-metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site-directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.
Amino Acid Sequence ; Base Sequence ; Calorimetry, Differential Scanning ; Catalytic Domain ; Crystallography, X-Ray ; DNA ; metabolism ; DNA Cleavage ; Deoxyribonucleases, Type II Site-Specific ; chemistry ; genetics ; metabolism ; Escherichia coli ; metabolism ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Recombinant Proteins ; chemistry ; genetics ; metabolism ; Sequence Alignment ; Substrate Specificity