A rapid method for preparation of plasmid DNA for screening recombinant clones / 生物工程学报

A simple and rapid method for preparation of plasmid DNA from overnight incubation was introduced. It does not require any additional reagents; the incubation mixture containing recombinant plasmid DNA was just mixed with H2O and phenol/chloroform/isoamyl alcohol in certain ratio. After vortexing and spinning of the mixture, the supernatant could be directly loaded onto agarose gel and analyzed using electrophoresis. The whole preparation requires only 3-5 minutes. So to quickly screen recombinant clones, this method is better compared with traditional methods.

Correlativity between the polymorphisms of aldosterone synthase gene, Hind III restriction site on Y chromosome and essential hypertension / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate the relationship of associating the polymorphisms of CYP11B2 -344C/T and Hind III restriction site on Y chromosome with essential hypertension.</p><p><b>METHODS</b>This study enrolled 654 patients with essential hypertension and 386 healthy subjects as control group. The genomic DNA was extracted from blood leukocytes. The DNA segments of CYP11B2 and Y chromosome were amplified from genomic DNA by polymerase chain reaction (PCR). The PCR products were digested with Hae III or Hind III at 37 degrees centigrade respectively. The digested products were subjected to agarose gel electrophoresis and stain with ethidium bromide.</p><p><b>RESULTS</b>(1)The Hind III (-) genotype was found at 42.0% for patients with essential hypertension and 32.9% for control. The Hind III (-) genotype frequency of hypertension patient was significantly higher than that of the control (P was 0.03). The Hind III (+) genotype had a lower SBP and DBP than the Hind III (-) genotype (P was 0.01, P was 0.03). (2)With combining CC or CT genotype with Hind III (-) genotype, the relative risk suffering from hypertension was 1.998 fold high (P was 0.01).</p><p><b>CONCLUSION</b>The polymorphism of Hind III restriction site on Y chromosome is associated with essential hypertension, and when combined with polymorphism of CYP11B2 -344C/T, may have a united role to increase the risk of suffering from hypertension disease.</p>

Uso del análisis de restricción en el control de calidad de cepa del biolarvicida cubano BACTIVEC / Use of the restriction analysis in the quality control of the strain of Cuban biolarvicide BACTIVEC

Se mostró la caracterización genómica de la cepa utilizada en la producción del biolarvicida Bactivec. Se compararon los patrones de digestión de la cepa de referencia 266 de Bacillus thuringiensis de la colección del Instituto Pushkin del antiguo Leningrado y 2 cepas aisladas de 2 lotes del biolarvicida. Las digestiones del ADN cromosomal se realizaron con las enzimas HindIII y EcoRI. En todos los casos se observó el mismo patrón de restricción, lo que muestra la estabilidad genética del ingrediente activo del biolarvicida Bactivec

Study on lipoprotein lipase gene Hind III polymorphism in Chinese type IIb hyperlipoproteinemia / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To investigate whether lipoprotein lipase (LPL) gene Hind III polymorphism is associated with Chinese type IIb hyperlipoproteinemia.</p><p><b>METHODS</b>Lipoprotein lipase gene Hind III polymorphism was studied using polymerase chain reaction (PCR) and restriction fragment length polymorphisms (RFLP) in 103 type IIb hyperlipoproteinemia patients and 129 healthy subjects from a population of Chinese Hans in Chengdu area.</p><p><b>RESULTS</b>Both in type IIb hyperlipoproteinemia group and control group, the H+H+ homozygote was the major allelotype. The H+ allelic frequency of type IIb hyperlipoproteinemia group was higher than that of control group (0.864 vs 0.705, P<0.01). But the H- allelic frequency of type IIb hyperlipoproteinemia group was significantly lower than that of control group (0.136 vs 0.295, P<0.01). The plasma triglycerides (TG) level of H+H+ genotype was significantly higher than that of H+H- and H-H- genotypes (P<0.05 and P<0.01); the plasma TC level and TG/HDL C ratio were higher than those of H+H- and H-H- genotypes (P<0.05); apoA II levels of H+H+ and H+H- genotypes were significantly lower than that of H-H- genotype (P<0.01 and P<0.05).</p><p><b>CONCLUSION</b>The Hind III RFLP at intron 8 of LPL gene is associated with type II b hyperlipoproteinemia to some extent in Chinese population.</p>

Cloning and polymorphism analysis of prtH gene from Porphyromonas gingivalis / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To clone the prtH gene from Porphyromonas gingivalis (P.g) ATCC 33277 and analyze the polymorphism of prtH gene from 5 strains of P.g in order to explore the relationship between P.g and periodontitis.</p><p><b>METHODS</b>Using PCR, the prtH was amplified and cloned into pGEM-T vector. To illustrate the prtH polymorphism among P.g strains, the genomic DNAs were extracted and screened by PCR with three pairs of specific primers, dot blot and Southern blot hybridization using the biotin-labeled prtH sequence as probe.</p><p><b>RESULTS</b>Recombinant DNA pGEM-T- prtH was verified by restriction endonuclease and sequence assay. Strain W 381 and ATCC 33277 showed the identical results in PCR and hybridization assays, whereas strain ATCC 49417 and 14-3-2 revealed individual hybridization patterns. Strain 47A-1 seemed even not to contain prtH gene.</p><p><b>CONCLUSIONS</b>Different prtH gene sequences exist in different P.g strains. This polymorphism may indicate various potential virulent effects during the infection and pathogenesis. Established PCR protocol is sensitive for identification of prtH gene.</p>

Purification and differentiation of three avian adenoviruses by restriction endonuclease analysis.

A rapid method of ultracentrifugation pelleting of avian adenovirus (AAV) from small volume of chloroform treated infected cell culture fluid or allantoic fluid was adapted for isolation of adenoviral DNA. The viral DNA extracted from semipurified viruses was found to be intact on agarose gel and pure enough (A260/280 = 1.85-1.92) for restriction enzyme analysis. Restriction endonuclease analysis of Indian strain of AAV serotype 1, AAV serotype 4 (group I AAVs) and egg drop syndrome-76 (EDS-76) virus genomes (group III AAV) with Hind III enzyme differentiated these viruses. The AAV serotype 1 and serotype 4 strain exhibited identical Hind III profile to European viral strains belonging to same serotypes however, the EDS-76 virus gave similar but not identical profile. The calculated genomic lengths for AAV serotype 1 and EDS-76 virus were approximately found to be 33.9 and 44.4 Kb, respectively.

Plasmid Profile and B - Lactamase Type of Multidrug - Resistant Salmonella typhi Isolated from Korea, 1997

Eight strains of multidrug-resistant (MDR) Salmonella typhi were isolated from Kyonggi area during January-February,1997. They were resistant to ampiciUin, amoxicillin, carbeniciillin, tetracycline, chloramphenicol, trimethoprim/sulfamethoxazole, trimethoprim. Eight strains had one plasmid respectively which size was approximately M.W 220 kb and showed same restriction pattern by endonuclease HindIII. The plasmid was similar to the plasmid in size that was related to multidrug resistant S. typhi isolated from southeast Asia. It were transferred by conjugation to recipient E, coli K-12 in frequency of 2.43 x10-4 - 1.73 x 10-2 and transconjugant showed same drug-resistant pattem with donor cells. All of 8 strains produced B-lactamase that was assummed to TEM-1 type by isoelectric focusing and PCR.

Polimorfismos del gen de lipoproteína lipasa en diabéticos no insulino-dependientes: estudio preliminar / Lipoprotein gene polymorphism in non dependent diabetics: preliminary study

Background: Lipoprotein lipase plays a crucial role in plasma lipoprotein metabolism. Several lipoprotein lipase gene polymorphisms have been found associated with lipid levels, premature atherosclerosis and cardiovascular disease. Aim: To investigate, in the Chilean population, the genotype distribution of lipoprotein lipase polymorphism and its possible association with lipid levels and obesity. Patients and methods: Hind III and Pvu II polymorphism was determined in 45 non-insulin-dependent diabetic patients and in 52 non diabetic controls from Santiago, Chile. Results: Hind III (+/+) polymorphism had a higher frequency in diabetics as compared to controls (0.6 and 0.29 respectively, p= 0.009). The frequency of heterozygous distribution was higher in non diabetic subjects. Controls and diabetics had comparable gene frequencies for the Pvu II genotype distribution. Analyzing the impact of these polymorphisms on plasma lipid levels, Hind III (+/+) genotype was associated with high Levels of total cholesterol and triglycerides in both groups. The hterozygote (+/-) or homozygote (-/-) state for Hind III was effectively associated with high levels of HDL cholesterol levels, as compared to the (+/+) genotype. There was no relationship between these genotypes and body mass index and waist to hip ratio. Conclusions: An association between genetic variation at the lipoprotein lipase locus with high levels of triglycerides and total cholesterol was confirmed. However, no association of these genetic markers with anthropometric measurements was found

Cloning of 2.4 kb bovine herpesvirus-1 DNA fragment containing glycoprotein III gene into pUC18 plasmid vector.

A recombinant pBR322 plasmid containing bovine herpesvirus-1 HindIII 'I' fragment was analysed using EcoRI and BamHI restriction endonucleases. This recombinant plasmid was labelled with [alpha 32P]dATP and hybridized with southern blot of HindIII digested BHV-1 DNA fragments. A 2.4 kb double digested EcoRI-BamHI fragment of HindIII 'I' was subcloned into pUC18 plasmid to get complete gIII gene. The recombinant pUC18 plasmid was analysed for 2.4 kb BHV-1 DNA insert by restriction digestion with EcoRI and BamHI. Southern blot of restriction digested plasmid was hybridized with [alpha 32P]dATP labelled BHV-1 DNA probe.

Detecçäo de portadoras de gene da hemofilia A em uma populaçäo brasileira / Carriers detection of hemophilia A gene in a Brazilian population

A identificaçäo de portadores do gene para a hemofilia A foi levada a efeito em 20 famílias de pacientes que apresentavam essa condiçäo. A detecçäo foi realizada com base no estudo dos polimorfismos de restriçäo (RLFP) do gene do fator VIII para as enzimas BcII e HindIII. Os métodos utilizados incluíram a amplificaçäo de fragmentos do gene em análise pela reaçäo em cadeia de polimerase e posterior digestäo com as enzimas específicas. Foram estudados 84 indivíduos pertencentes a 20 famílias de pacientes. Foi possível identificar ou excluir portadoras do gene anormal em 76// das famílias estudadas. Esse traballho evidencia a possibilidade de detecçäo de portadoras na maioria das famílias de pacientes com hemofilia A na populaçäo brasileira com o emprego de um número restrito de polimorfismo de restriçäo

Molecular cloning and restriction endonuclease analysis of 0.4 kb Hin dIII'O' fragment of bovine herpesvirus 1 DNA.

Bovine herpesvirus 1 DNA has been isolated by SDS lysis of the virus purified from potassium tartrate (10-50%) density gradient centrifugation. The quality and quantity of viral DNA was checked by UV spectrophotometry and ethidium bromide stained agarose gel electrophoresis. The 0.4 kb Hin dIII'O' fragment of BHV-1 DNA was selectively cloned into Hin dIII cut pUC9 plasmid DNA (2.665 kb). Recombinants were screened by white/blue colonies as well as Hin dIII restriction enzyme analysis. On restriction endonuclease analysis of recombinant plasmid DNA (p-BH-0) with several restriction enzymes, viz., Sau 3A, Hin fI, Rsa I, Sal I, Dra I, Bgl I, Bgl II, Sma I, Hpa I, Stu I, Mlu I, Xho I, Kpn I, Hae III, Eco RI, Bam HI, Pst I, Pal I, revealed insert viral DNA having sites for Hin fI, Hae III, Rsa I, Sma I, only. Further, the partial restriction map of the recombinant plasmid DNA was constructed using above enzymes.