Oliveira Caio Fernando de, Paim Thiago Galvão da Silva, Reiter Keli Cristine, Rieger Alexandre, D'Azevedo Pedro Alves
Health Sciences Post-graduate Program, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA), Health Sciences Post-graduate Program, Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA).
Laboratory of Biotechnology and Genetic, Universidade de Santa Cruz do Sul (UNISC), Laboratory of Biotechnology and Genetic, Universidade de Santa Cruz do Sul (UNISC).
Rev Inst Med Trop Sao Paulo. 2014 Jan-Feb;56(1):29-33. doi: 10.1590/S0036-46652014000100004.
Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.
目前，基于不同原理有几种提取细菌DNA的方法。然而，这些方法中每种方法所获得的DNA的量和质量差异很大，并且取决于微生物，例如凝固酶阴性葡萄球菌（CoNS），其具有难以裂解的厚细胞壁。本研究旨在比较通过四种不同技术提取的CoNS DNA的质量和量：两种内部方案和两种商业试剂盒。通过分光光度法进行DNA量和质量的测定。还使用琼脂糖凝胶电泳和PCR对提取的DNA进行分析。本研究使用了267株CoNS分离株。柱法和热裂解在DNA质量（A260/280平均比值 = 1.95）和DNA平均浓度方面分别显示出更好的结果。所测试的所有四种方法都提供了适用于PCR扩增的DNA，但产量不同。DNA质量很重要，因为它允许应用大量分子生物学技术，并且还能长时间储存。从这个意义上讲，基于提取柱的提取方法对CoNS显示出最佳结果。
