ABSTRACT
Objective To optimize the extraction methods of mitochondrial genome DNA(mtDNA)of Oncomelania hupen-sis. Methods The pyrolysis,protein K variable-temperature digestion and high-concentration potassium acetate purification were applied to optimize the high-concentration-salt precipitation method,and then the optimized method was compared with two common extraction methods,the sucrose density gradient centrifugation method and traditional high-concentration-salt pre-cipitation method. The mtDNA samples were identified by using spectrophotometry,agarose gel electrophoresis and the amplifi-cation products of COX1. The nuclear DNA contamination was tested by the amplification products of ITS. Results The concen-tration and yield of the improved method was significantly higher than those of the traditional method(F=3032.65,10185.00, both P<0.01). The mtDNA samples extracted were essentially free of nuclear DNA and protein,meeting PCR,sequence analy-sis and other molecular biology research requirements. Conclusions The improved high-concentration-salt precipitation meth-od for isolating mtDNA is simple,and it has high yield and low cost. The extracted mtDNA can meet relevant analysis require-ments.
ABSTRACT
Objective To establish a multiplex polymerase chain reaction (M-PCR) assay to detect HBV DNA in the peripheral blood mononuclear cells(PBMCs) of chronic HB patients. Methods One pair of primer amplifying HBV genome DNA and another pair of primer amplifying HBV covalently closed circular DNA (cccDNA ) were added to one PCR reaction to detect HBV DNA in PBMCs. Results Various forms of HBVDNA including total DNA and cccDNA could be amplified simultaneously. Among the 30 chronic HB patients, both the HBVDNA and HBVcccDNA in the PBMCs of 23 patients were detected, the positive rate of which was 76.6%. The positive rate of HBV cccDNA accounted for 82.1% of total HBV DNA positive rate. Conclusion HBVDNA in the PBMCs could partially replicate. The M-PCR was successfully set up to amplify HBV genome DNA and HBV cccDNA simultaneously.
ABSTRACT
Objective With the sole object of glycyrrhizic acid products,the methods were investigated for Glycyrrhiza uralensis genomic DNA transformation into Hansenula anomala by nitrogen and argon ion implantation.Methods The genomic DNA from G.uralensis was randomly transferred into H.anomala by nitrogen and argon ion bombardment.The recombined yeasts were cultured by the slant and liquid cultivation,in which the contents of glycyrrhizic acid and glycyrrhetic acid were determined by acetic anhydried-H2SO4 qualitative test and RP-HPLC determination.Results Five recombined yeast strains that produced glycyrrhizic acid and glycyrrhetic acid were obtained.After cultured in liquid medium for 96 h,the highest content of glycyrrhizic acid in the cultivation liquid was 114.49 mg/L and that of 18?-glycyrrhetic acid and 18?-glycyrrhetic acid were respectively 0.56 and 0.81 mg/L by RP-HPLC.A kind of unknown red component was found in the cultivation liquid of one recombined strain by TLC.Conclusion The recombined yeast strains of producing glycyrrhizic acid could be obtained G.uralensis genomic DNA transformation into yeast mediated by the ion implantation.
ABSTRACT
Objective To develop a whole-genome DNA microarray based on the genomic sequences of Y. pestis CO92 and 91001 and its use in comparative genomic analysis. Methods A total number of 4 005 genes of Y. pestis were amplified by PCR and printed onto glass slides in duplicate. Fluorescently labeled probes were prepared by marking genomic DNAs with random hexamers and Klenow. Labeled DNAs were hybridized with the microarrays by the method of two-fluorescence comparative hybridization. Three sets of two-fluorescence hybridizations were performed to examine the absence/presence of each gene. Results The results agreed with those derived from the in silico genomic comparison. Conclusion The results demonstrate that the microarry can be a useful tool for comparative genomic analysis of Y. pestis.