The extraction of high-molecular-mass DNA from hair shafts

A simple and efficient method is presented for the extraction of DNA from hair shafts. DNA preparations obtained by this approach can be made amenable to restriction enzyme digestion, thereby allowing further molecular biological analysis.     

模式小鼠总DNA三种提取方法比较

目的：建立简便、快捷、经济的模式小鼠总DNA提取方法,以快速鉴定大批量模式小鼠基因型。方法采用苯酚抽提法、异丙醇沉淀法、鼠耳煮沸法提取同种模式小鼠总DNA,对比DNA纯度、得率、耗费时间,并比较基因型鉴定结果。结果苯酚抽提法得率最高,异丙醇沉淀法最低;而纯度则按照苯酚抽提法、异丙醇沉淀法、鼠耳煮沸法顺序递减;在耗时上鼠耳煮沸法最短。三种方法提取的DNA均可做模版用于基因型鉴定。结论鼠耳煮沸法操作简单、成本最低,快速、基因型鉴定结果可靠,可用于规模化的基因型鉴定实验中。     

Comparison of rapid methods for the extraction of bacterial DNA from colonic and caecal lumen contents of the pig

AIMS: The increasing uses of DNA methodologies to study the micro flora of the pig gastrointestinal tract requires an efficient recovery of bacterial DNA from the intestinal sample. Thus, the objective of this study was to determine which DNA extraction methods are most effective for luminal samples from pigs. Several routinely used nucleic acid extraction procedures were compared based upon quantity and purity of extracted DNA. METHODS AND RESULTS: DNA was extracted from pig colonic and caecal lumen samples using 19 methods for bacterial DNA extraction. The quantity of total DNA recovered by each extraction method was determined and compared. Two methods using extraction with polyvinylpolypyrrolidone (PVPP) or phenol and two methods involving bead mill homogenization were found to provide the greatest quantity of extracted DNA for both colonic and caecal lumen. Extracted DNA from these four methods was further analysed for purity based upon the presence of PCR inhibitors, which was ascertained by determining the efficiency of amplification of a segment of the 16S rDNA. PCR amplification could be readily achieved with DNA extracted by each of these four methods, but efficiency of amplification tended to be higher with DNA from two of the methods (one extracted with PVPP and one with bead mill homogenization). CONCLUSIONS: Four extraction methods proved to be significantly superior in quantity of DNA extracted from luminal samples. Of these four, no strong inhibitors of PCR amplification were detected in any of the extracted DNA. However, the efficiency of amplification tended to be lower in DNA samples from two of the methods, suggesting the presence of low levels of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: Results of this study provide a basis for choosing which DNA extraction procedures are most effective for use with samples of pig lumen.     

DNA extraction for 16S rRNA gene analysis to determine genetic diversity in deep sediment communities

A protocol was devised which permitted the extraction of DNA from deep marine sediments up to 503 m below the sea floor. These sediments have been laid down over the last 3 million years. 16S rRNA gene sequences were amplified from the DNA by the polymerase chain reaction. The details of the successful extraction and polymerase chain reaction methodology varied between samples from different depths. This emphasizes the attention to detail required to allow the diversity of bacteria in these deep sediments to be studied.     

Integrated method for single-cell DNA extraction, PCR amplification, and sequencing of ribosomal DNA from harmful dinoflagellates Cochlodinium polykrikoides and Alexandrium catenella

A simplified technique was developed for DNA sequence-based diagnosis of harmful dinoflagellate species. This protocol integrates procedures for DNA extraction and polymerase chain reaction (PCR) amplification into a single tube. DNA sequencing reactions were performed directly, using unpurified PCR products as the DNA template for subsequent sequencing reactions. PCR reactions using DNA extracted from single cells of Cocodinium polykrikoides and Alexandrium catenella successfully amplified the target ribosomal DNA regions. DNA sequencing of the unpurified PCR products showed that DNA sequences corresponded to the expected locus of ribosomal DNA regions of both A. catenella and C. polykrikoides (each zero genetic distance and 100% sequence similarity). Using the protocol described in this article, there was little DNA loss during the purification step, and the technique was found to be rapid and inexpensive. This protocol clearly resolves the taxonomic ambiguities of closely related algal species (such as Alexandrium and Cochlodinium), and it constitutes a significant breakthrough for the molecular analysis of nonculturable dinoflagellate species.     

Evaluation of DNA extraction methods from complex phototrophic biofilms

Phototrophic biofilms are used in a variety of biotechnological and industrial processes. Understanding their structure, ie microbial composition, is a necessary step for understanding their function and, ultimately, for the success of their application. DNA analysis methods can be used to obtain information on the taxonomic composition and relative abundance of the biofilm members. The potential bias introduced by DNA extraction methods in the study of the diversity of a complex phototrophic sulfide-oxidizing biofilm was examined. The efficiency of eight different DNA extraction methods combining physical, mechanical and chemical procedures was assessed. Methods were compared in terms of extraction efficiency, measured by DNA quantification, and detectable diversity (16S rRNA genes recovered), evaluated by denaturing gradient gel electrophoresis (DGGE). Significant differences were found in DNA yields ranging from 116 ± 12 to 1893 ± 96 ng of DNA. The different DGGE fingerprints ranged from 7 to 12 bands. Methods including phenol–chloroform extraction after enzymatic lysis resulted in the greatest DNA yields and detectable diversity. Additionally, two methods showing similar yields and retrieved diversity were compared by cloning and sequencing. Clones belonging to members of the Alpha-, Beta- and Gamma- proteobacteria, Bacteroidetes, Cyanobacteria and to the Firmicutes were recovered from both libraries. However, when bead-beating was applied, clones belonging to the Deltaproteobacteria were also recovered, as well as plastid signatures. Phenol–chloroform extraction after bead-beating and enzymatic lysis was therefore considered to be the most suitable method for DNA extraction from such highly diverse phototrophic biofilms.     

鸡肠道菌群总DNA三种提取方法的比较

目的比较不同方法提取鸡肠道菌群总DNA的差异,为分子方法分析肠道菌群组成提供质量较高的DNA模板。方法采用反复冻融法、酶裂解法和试剂盒法（E.N.Z.A Stool DNA Kit）来提取鸡肠道菌群的总DNA,并根据DNA浓度及纯度、16S DNA扩增产物和ERIC-PCR产物所反映的片段多态性4个指标,对这3种方法提取的DNA质量进行比较。结果3种方法均能提取DNA,所得DNA都可以用于16S DNA的扩增,但后2种方法所得DNA的ERIC-PCR结果能反映出更高的菌群多样性。结论试剂盒法和酶裂解法所提取的DNA质量好,适合用于肠道菌群的分子生态研究。     

两种提取肠道微生物总DNA方法的比较

目的比较肠道微生物总DNA两种提取方法的优缺点。方法采用苯酚-氯仿抽提法和试剂盒法提取肠道微生物总DNA,比较其作为模板扩增细菌16S DNA的优缺点。结果两种方法提取的细菌DNA均能用于后续PCR扩增的模板。酚-氯仿抽提法经济可靠,但提取的DNA量及纯度较低试;剂盒法提取方便,操作简单,质量稳定,易标准化,但成本高。结论两种方法各具优缺点,应根据实验室条件和实验要求进行选择。     

Microwave-assisted digestion combined with silica-based spin column for DNA isolation from human bones

A protocol for the extraction of DNA from ancient skeletal material was developed. Bone specimen samples (powder or slice), buffer, pretreatment, and extraction methodologies were compared to investigate the best conditions yielding the highest concentration of DNA. The degree of extract contamination by polymerase chain reaction (PCR) inhibitors was compared as well. Pretreatment was carried out using agitation in an incubator shaker and microwave digestion. Subsequently, DNA from bones was isolated by the classical organic phenol–chloroform extraction and silica-based spin columns. Decalcification buffer for total demineralization was required as well as lysis buffer for cell lysis to obtain DNA, whereas microwave-assisted digestion proved to be very rapid, with an incubation time of 2 min instead of 24 h at an incubator shaker without using lysis buffer. The correction of isolated DNA was detected using real-time PCR with melt curve analysis, which was 82.8 ± 0.2 °C for highly repetitive α-satellite gene region specific for human chromosome 17 (locus D17Z1). Consequently, microwave-based DNA digestion followed by silica column yielded a high-purity DNA with a concentration of 19.40 ng/μl and proved to be a superior alternative to the phenol–chloroform method, presenting an environmentally friendly and efficient technique for DNA extraction.     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.     

三种提取蓝猪耳总DNA方法的比较

针对蓝猪耳（Torenia fournieri L.）叶片中多糖、色素等物质严重干扰总DNA提取质量的问题,以蓝猪耳叶片为试验材料,分别采用高盐低pH值法、SDS法、CTAB法提取总DNA,并从样品电泳图谱、纯度、得率等方面对三种方法进行评价。结果表明,CTAB法提取总DNA的产率较高、纯度最好,是蓝猪耳总DNA提取的最佳方法。     

4种黑曲霉基因组DNA提取方法的比较

目的:筛选适合提取曲霉DNA的方法.方法:比较2个菌落培养时间段(3d内和10d左右)提取DNA质量的差异;运用氯化苄法、石英砂+CTAB法、Biospin法和微波法分别提取黑曲霉基因组DNA,然后用直接电泳、浓度测定、PCR扩增等方法比较所提DNA的浓度和质量.结果:培养3d内的菌落提取的DNA纯度较高,无需纯化即可用于后续实验;4种方法制备的DNA均可用于PCR等后续实验,其中以石英砂+CTAB法提取的DNA纯度好,产率最高.结论石英砂+CTAB法是一种适用于曲霉DNA提取的简便方法.     

Plasmid and chromosomal DNA recovery by electroextraction of cyanobacteria

Abstract High voltage electroporation has been investigated as a method for rapid recovery of plasmid and chromosomal DNA from the cyanobacteria Nostoc PCC 7121, Synechococcus PCC 7002, and Anabaena PCC 7120. Pulses of 18 kV/cm and higher applied to concentrated Nostoc cells carrying a shuttle plasmid (pRL25) resulted in copious release of nucleic acids and phycobiliproteins into the suspending medium. Small portions of these supernatants, when electroporated with Escherichia coli , gave rise to hundreds of E. coli transformants which contained pRL25. Electroporation of Synechococcus carrying plasmid pAQE19 did not cause detectable release of macromolecules but did reveal a low-level, voltage independent 'leakage' of pAQE19 into the medium. Electroextraction of Nostoc or Anabaena followed by addition of E. coli and delivery of a second high-voltage pulse permitted direct, one-cuvette transfer of shuttle plasmids from these cyanobacteria into E. coli . Electroextraction of single cyanobacterial colonies, as shown for Nostoc , also released sufficient chromosomal DNA for amplification of specific sequences by the polymerase chain reaction.     

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples

Aims

Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10‐fold serial dilutions of Bacillus anthracis spores using quantitative real‐time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10¹ and 1·3 × 10² CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS).

Methods and Results

The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors.

Conclusions

Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit.

Significance and Impact of the Study

The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.     

Comparison of three methods of DNA extraction from cold-smoked salmon and impact of physical treatments

AIMS: To compare three bacterial DNA extraction procedures on cold-smoked salmon (CSS) and assess the impact on their efficiency of two physical treatments of the food matrix, ionizing irradiation and freezing. METHODS AND RESULTS: As molecular methods for bacterial detection have become an important analytical tool, we compared bacterial DNA extraction procedures on CSS. Working with frozen and irradiated CSS, we obtained negative responses from samples known to be highly contaminated. Thus, we decided to study the impact of these two physical treatments on bacterial DNA extraction procedures. The efficiency of bacterial DNA extraction directly from the fish matrix suspension was measured by an rpoB PCR-based reaction. The results demonstrated that the DNeasy tissue extraction kit (Qiagen, Courtaboeuf, France) was the most efficient and reproducible method. We also showed that freezing and ionizing irradiation have a negative impact on DNA extraction. This was found probably not to be due to inhibition as the PCR reaction remained negative after adding BSA to the PCR mix reaction. CONCLUSIONS: The extraction kit was the most efficient method. Physical treatments were shown to hamper bacterial DNA extraction. SIGNIFICANCE AND IMPACT OF THE STUDY: Attention must be paid to molecular bacterial detection on food products subject to freezing or to ionizing irradiation.     

Recombinant DNA probes and polymerase chain reaction for detection of Mycoplasma gallisepticum strains

Abstract Two recombinant DNA clones, pMG286.2 and pMG301.1, were isolated from the partial genomic library of Mycoplasma gallisepticum strain S6. Recombinant M. gallisepticum specific fragments were used as probes in Southern hybridisation with 10 M. gallisepticum strains whose DNA was digested by Eco RI, Hin dIII, Bgl II, Rsa I and Bam HI. The 1.5 kb fragment pMG301.1 did not show polymorphism in hybridisation patterns with M. gallisepticum strains, while the 3.5 kb fragment pMG286.2 enabled differentiation of M. gallisepticum strains into clusters. The DNA sequence of pMG301.1 was used to design a pair of 27-mer oligonucleotides flanking a 1.3 kb genomic region. These two primers directed specific in vitro amplification of all M. gallisepticum strains assayed giving an expected 1.3 kb product. Digestion of polymerase chain reaction products by Dde I enabled simple differentiation between clusters of M. gallisepticum strains and may be useful for improved epizootiological studies of M. gallisepticum infections in poultry.     

利用改进的酚-氯仿法从猪毛囊中提取基因组DNA

为提高从猪毛囊组织中提取基因组DNA的效率, 文章在借鉴从其他组织提取基因组DNA方法的基础上, 对经典的酚-氯仿法的反应体系和步骤进行了改进。利用改进的酚－氯仿抽提法, 从猪的毛囊组织中快速、高效地提取了高质量基因组DNA。利用该方法从1~6根猪毛囊中提取的基因组DNA可满足基于PCR技术的相关分子生物学实验需要。     

红豆杉属植物三种不同总DNA提取方法的分析比较

红豆杉属植物均为濒危物种,也是国家一级保护植物.以红豆杉属植物叶片为材料,利用三种不同的DNA提取方法提取总DNA,用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的得率和纯度,用PCR扩增的方法检测所得总DNA的质量,并对三种不同提取方法的结果进行了比较分析.结果表明:CTAB法提取的DNA纯度和得率均较高,可直接用...     

Optimal extraction methods for the simultaneous analysis of DNA from diverse organisms and sample types

Using environmental DNA (eDNA) to assess the distribution of micro‐ and macroorganisms is becoming increasingly popular. However, the comparability and reliability of these studies is not well understood as we lack evidence on how different DNA extraction methods affect the detection of different organisms, and how this varies among sample types. Our aim was to quantify biases associated with six DNA extraction methods and identify one which is optimal for eDNA research targeting multiple organisms and sample types. We assessed each methods’ ability to simultaneously extract bacterial, fungal, plant, animal and fish DNA from soil, leaf litter, stream water, stream sediment, stream biofilm and kick‐net samples, as well as from mock communities. Method choice affected alpha‐diversity for several combinations of taxon and sample type, with the majority of the differences occurring in the bacterial communities. While a single method performed optimally for the extraction of DNA from bacterial, fungal and plant mock communities, different methods performed best for invertebrate and fish mock communities. The consistency of methods, as measured by the similarity of community compositions resulting from replicate extractions, varied and was lowest for the animal communities. Collectively, these data provide the first comprehensive assessment of the biases associated with DNA extraction for both different sample types and taxa types, allowing us to identify DNeasy PowerSoil as a universal DNA extraction method. The adoption of standardized approaches for eDNA extraction will ensure that results can be more reliably compared, and biases quantified, thereby advancing eDNA as an ecological research tool.     

Comparison of DNA extraction methods for human gut microbial community profiling

The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome.