ABSTRACT
Background: DNA extraction has become a baseline method for molecular biology studies. There are a variety of methods available for this purpose. Newer kit-based methods (KBM) are easy and less time consuming than traditional chemical methods of extraction like phenol chloroform method (PCM). Though estimates of quality from different methods are available in labels, this study compared the practical outcomes regarding quantity, quality, DNA recovery rate and assessed the outcomes at two different time points.Methods: This study was done as a secondary analysis from an ongoing project. The quantity and quality of DNA isolated from the same group of 100 deidentified blood samples by PCM and KBM were analysed using Multi analyzer and repeated after a period of 3 months. Genotyping of the samples were done by RT-PCR. The quantity, quality and amplification proportion were compared between two groups to reach the inference.Results: The median (range) concentration of DNA by PCM was 543.27 (960.59) µg/ml and that of KBM was 32.115 (36.73) µg/ml. The quality of DNA as measured by absorbance at 260/280 nm was 1.84 in PCM and 1.81 in KBM (p>0.05). Genotyping success rate was 78% in PCM and 98% in KBM (p = 0.002). The DNA recovery rate was 96% in PCM and 80% in KBM (p=0.014).Conclusions: The median concentration of DNA obtained from PCM was more compared to KBM. The quality of DNA was comparable in both the groups. The genotyping success rate was more in KBM group. The DNA recovery rate at 3 months was more in PCM group.
ABSTRACT
Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (P<0.01). PCR results revealed that method A was more reliable in amplifying DEAD-box polypeptide 4 (DDX4) and copy number variations (CNVs) than methods B and C, which generated false-positive errors. The results of sperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.
Subject(s)
Adult , Humans , Male , DNA , Metabolism , DNA Methylation , Electrophoresis, Agar Gel , Spermatozoa , MetabolismABSTRACT
BACKGROUND: Vibrio vulnificus is a pathogenic, marine, halophilic, gram-negative bacillus which causes fulminant infecticn in humans through the ingestion of raw seafood or skin wounds. V. vulnificus produces seveal kinds of virulent factors including cytolysin, endotoxin, exoenzymes, and siderphores. Among these, the lipopolysaccharide(LPS) of V. vulnificus has recently been purified, but the biological activity of this endotoxin is not well clarified as yet. OBJECTIVE: The purpose of this study was to extract LPS from Vibrio vulnificus and to test the biological activity of extracted LPS for the elucidation of the role in V. vulnificas septicemia. METHODS: V. vulnificus LPS was extracted by the Phenol Chloroform-Petroleurn ether(PCP) method. The biological activity of LPS was evaluated with a limulus amebocyte lysate assay and by assessment of lethality to ICR mice. RESULTS: Five hundreds mg of LPS were extracted from 10g of dried V. vulnificus. Lirnulus amebocyte lysate formed a hard gel in response to the extracted LPS. This LPS showed low level of protein contaminatior in SDS-PAGE electophoresis and spectrophotometry. A High dose of LPS(200 mg/ml body weight) was lethal to mice. CONCLUSION: The PCP extraction yield relatively large amounts of LPS from V. vnlnificus. with out significant protein contamination and e xtrated LPS has endotoxin activity. This extrated LPS can be used for further studies such as making antibody or characterizing pathogenic roles in the V. vulnificus infection.