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OBJECTIVE: To extract microamounts of animal-derived DNA from the products of Colla Corii Asini boiled at high temperature, establish and optimize a rapid identification method of donkey-derived components in Colla Corii Asini by polymerase chain reaction(PCR), and establish a new molecular biological method for assessing the quality of Colla Corii Asini. METHODS: The donkey-derived genomic DNA was extracted by DNA purification column instead of phenol, chloroform and other toxic organic solvents in SDS-PK method, and the SDS-PK method was optimized with the donkey-derived genomic DNA. RESULTS: The optimum sample size of Colla Corii Asini was 0.20 g. High quality genomic DNA of Colla Corii Asini could be obtained quickly after digestion in water bath for 1 h and then purified by DNA purification column. The purity ranged from 1.70 to 1.80, and the concentration of Colla Corii Asini could reach (187.8±0.56)ng•μL-1. PCR amplification, cloning, and sequencing were performed using specific primers, and the similarity to GenBank's registered Donkey species (MG931481.1) was 100%. CONCLUSION: This study provides animal-derived genomic DNA fragments from deep-processed Colla Corii Asini and Colla Corii Asini products within 90 min. The purity and concentration of extracted DNA can meet the requirements of molecular biological identification of Colla Corii Asini. The established PCR method can quickly identify the scorpion-derived components in Colla Corii Asini. The cloned donkey specific gene fragment can be used as a standard positive control to identify the authenticity of Colla Corii Asini. It is expected that it will be widely used in the quality supervision of Colla Corii Asini and related products.
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Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content.
Subject(s)
Centrifugation , DNA , DNA, Bacterial , Glass , Humic Substances , Magnesium , Methods , Polymerase Chain Reaction , Population Characteristics , SoilABSTRACT
Objective To study DNA quantification and STR typing of samples pre-treated with pyrami-don. Methods The blood samples of ten unrelated individuals were anticoagulated in EDTA. The blood stains were made on the filter paper. The experimental groups were divided into six groups in accor-dance with the storage time, 30 min, 1 h, 3 h, 6 h, 12 h and 24 h after pre-treated with pyramidon. DNA was extracted by three methods: magnetic bead-based extraction, QIAcube DNA purification method and Chelex-100 method. The quantification of DNA was made by fluorescent quantitative PCR. STR typing was detected by PCR-STR fluorescent technology. Results In the same DNA extraction method, the sample DNA decreased gradually with times after pre-treatment with pyramidon. In the same storage time, the DNA quantification in different extraction methods had significant differences. Sixteen loci DNA typing were detected in 90.56% of samples. Conclusion Pyramidon pre-treatment could cause DNA degradation, but effective STR typing can be achieved within 24 h. The magnetic bead-based extrac-tion is the best method for STR profiling and DNA extraction.
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Objective To elucidate physiological functions of human PIF1 helicase at the molecular level,purify N-terminal truncated PIF1 helicase,PIF1△N,and assay its biochemical properties.Methods The N-terminal cDNA sequence of PIF1 helicase was amplified by PCR using the Hela cell cDNA library as template.The cDNA with a histidine tag at the N-terminus was inserted into the pET20b vector to produce recombinant plasmid.The recombinant PIF1△N was successfully expressed by co-transforming a plasmid encoding rare rRNA.At 4 ℃ through a series of affinity column the recombinant PIF1△N protein was purified by fast protein liquid chromatograph.The biochemical activity of PIF1△N was assayed.Results The cDNA fragment of human PIF1 from 540～1 923 was cloned from Hela cDNA library,and the recombinant PIF1△N protein was successfully overexpressed in E.coli.The purification procedure of PIF1△N protein was established and its biochemical activity was identified.Conclusion N-terminal truncated PIF1 helicase,PIF1△N,has ATPase activity,which is DNA and Mg2+ dependent.