A simple method of DNA extraction from whole tissues and blood using glass powder for detection of transgenic animals by PCR

A very simple and reliable method to extract DNA directly from mouse tail, rabbit ear and blood is described. Tissue was homogenized in a solution of NaI and the DNA was extracted using glass powder. The extracted DNA was obtained in sufficient quantity and purity to allow direct detection of transgene by PCR.     

An inexpensive high-throughput method to extract high yields of good quality DNA from fungi

We developed an efficient method for high-throughput extraction of high-quality DNA from various fungi. In this method, fungal mycelia were cultured and harvested on the surfaces of membranes on media plates. We degraded cell walls using a lytic enzyme (Yatalase). Purification was performed on 96-well glass fibre filter plates. DNA was successfully extracted from various fungi provided (102 genus 132 species) at high yields and quality, and proved suitable for storage, polymerase chain reaction amplification and restriction enzyme digestion. The method described is rapid, inexpensive and automation friendly. This enables the simultaneous extraction of large numbers of samples, significantly improving the potential throughput in genomics, particularly in diagnostic and population studies.     

Use of DNA from dry leaves for PCR and RAPD analysis

Fresh or frozen tissue is usually used as a source of DNA for PCR and RAPD analysis. We have found that leaves can be allowed to dry at room temperature before extraction of DNA. Heating the leaves or microwave drying resulted in poor recovery of DNA. Storage of fresh leaves in paper envelopes in the laboratory was the most successful approach. This allowed the tissue to dry out over a period of several days and DNA could be extracted at any time, providing a convenient method for the collection and analysis of field material. DNA from leaves stored for four months at room temperature was suitable for PCR analysis.     

一种快速提取肠道微生物总DNA的方法

采集的兔肠道内容物及其粪便样品,通过分散浸泡、震荡洗涤、分级离心、滤器过滤、DNA提取试剂盒提取纯化,可以获得纯度很高的DNA样品。经0.8%琼脂糖凝胶电泳检测和紫外分光光度计测定,样品A260/A280的比值为1.72±0.02。分别以提取的DNA样品为模板,通过设计的细菌特异引物,对其16S rDNA基因进行PCR扩增,获得了1.6 kb大小特异性很好的预期条带。这为肠道微生物群落的分子生态学研究提供了一种简便、可靠的DNA提取方法。     

Factors affecting mitochondrial DNA quality from museum preserved Drosophila simulans

In this study we investigate how traditional killing methods and storage regimes affected mitochondrial DNA quality in Drosophila simulans. Here we define quality with three criteria: (1) size of extracted DNA, (2) extraction yield, and (3) ability to amplify from four target regions. Killing methods had a significant effect on extraction yield, but not on PCR success. Highest DNA yields were extracted from specimens exposed to cyanide, while the lowest were from specimens killed in 70% ethanol. Specimens stored for two years contained badly sheared DNA, which translated into a significant decrease in PCR success compared to freshly assayed specimens. The most dramatic decrease in PCR success occurred in the 1822 bp and 1332 bp amplicons, compared to the 959 bp and 291 bp fragments. Naphthalene did not affect any aspect of DNA quality; time of storage affected PCR success regardless of naphthalene environment. This study serves to further refine our understanding of DNA degradation.     

Single Fish Egg DNA Extraction for PCR Amplification

Modern stock researches on marine biomass are basically genetic and rely increasingly on PCR-based manipulations of informative DNA markers for detecting the genetic diversity. This study developed a simple and rapid single tube method for DNA extraction from a single fish egg. The 15 min protocol was based on the use of Chelex 100 resin and urea to breakdown membrane and connective tissue of eggs. From various sizes of a single egg of walleye pollack (Theragra chalcogramma), the amounts of total nucleic acids were reproducibly obtained to be 18.25 ± 1.92 μg per egg. Using DNA templates diluted ranging 1/10⁰–1/10⁵, PCR amplification for the mitochondrial cytochrome b (Cytb) gene was successfully performed, and the 1/10² diluted template yielded the best result in PCR amplification for three different DNA marker genes. This method is quite simple and economical, and enables to provide the high throughput often demanded by the stock identification of marine biomass, in which large numbers of specimens of single fish eggs must be analyzed.     

A simple method of DNA extraction from soil for detection of composite transgenic plants by PCR

This new and simple method of DNA extraction from composite soil allows the isolation of plant DNA with high efficiency, quality and reproductivity. The method is based on a simple CaCl₂-precipitation step and requires no additional purification steps to eliminate humic acids. The extracted DNA was obtained in sufficient purity and quantity to allow direct detection of transgenes by PCR. Furthermore, the simple procedure allows the assay of many samples at the same time.     

活性污泥总DNA提取方法的研究

采用冻融＋玻璃珠、溶菌酶＋SDS和冻融＋玻璃珠＋溶菌酶＋SDS的方法提取活性污泥的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA。经紫外光度分析及电泳结果表明,冻融＋玻璃珠＋溶菌酶＋SDS方法所得的DNA的OD260/OD280为1．81,电泳结果显示,所提DNA片段分子量大于10kb,适于酶解和PCR扩增要求,为PCR技术应用于活性污泥的研究提供了一种简便、可靠的DNA提取方法。     

用于PCR扩增的DNA简易制备法现状

介绍了 6种用于PCR扩增的DNA简易制备法。所得DNA质量符合PCR扩增之用 ,经过试验也可用于系统生物学之研究     

一种冬虫夏草全基因组DNA的提取方法

冬虫夏草是真菌与昆虫形成的复合生物体,本研究建立了一种可同时提取冬虫夏草真菌子座和虫体全部基因组DNA的方法。该方法稳定高效,简便易行,提取纯度高,适用于冬虫夏草多重PCR、Realtime-PCR和DNA指纹图谱等分子水平的研究。     

对中国黄牛毛囊DNA六种提取方法的研究

本研究的目的是寻求一种简便、高效和对黄牛没有创伤的DNA提取方法,从而为黄牛基因组DNA的制备提供最优可行方案.本研究以中国黄牛毛囊作为DNA的提取材料,设计了离心柱法、磁珠法、SDS法、CTAB法、碱裂解法和煮沸法等6种DNA提取方法,然后分别对提取的DNA样本进行了分光光度计鉴定、PCR扩增和琼脂糖凝胶电泳.发现在...     

DNA Extraction from small blood volumes and the processing of cellulose blood cards for use in polymerase chain reaction

This article describes two procedures for the purification of genomic DNA from small blood volumes of whole blood using DNAzol^®BD. In the first procedure, DNA is isolated from 1–20 μL of whole blood using a fast and simple protocol that is appropriate for the simultaneous extraction of a large number of samples. The isolated DNA is suitable for gel electrophoresis and polymerase chain reaction (PCR). In the second procedure, cellulose blood cards containing approx 5 μL of dried blood are treated with DNAzol BD in order to retain DNA on the cellulose matrix while removing other cellular components. The blood card with DNA subsequently serves as template in PCR. The blood card processing and amplification procedures are performed in the same PCR tube without any centrifugation steps, making the combined procedures amenable for automated DNA preparation and amplification in a single tube.     

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of totalgenomic DNA from 15 red algal species. It resulted in 0.1 g high quality DNAfrom 1 mg fresh algal material, with an A₂₆₀/A₂₈₀ratio of 1.68–1.90.Using this rapidly isolated DNA, the 18S ribosomal RNA genes (rDNA) and the nuclearribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. Thetested DNA was suitable for restriction endonuclease digestion, genetic markeranalysis and polymerase chain reaction (PCR) amplification, and may be valid forother genetic manipulation.     

A simple method of genomic DNA extraction suitable for analysis of bulk fungal strains

Aims: A simple and rapid method (designated thermolysis) for extracting genomic DNA from bulk fungal strains was described. Methods and Results: In the thermolysis method, a few mycelia or yeast cells were first rinsed with pure water to remove potential PCR inhibitors and then incubated in a lysis buffer at 85°C to break down cell walls and membranes. This method was used to extract genomic DNA from large numbers of fungal strains (more than 92 species, 35 genera of three phyla) isolated from different sections of natural Ophiocordyceps sinensis specimens. Regions of interest from high as well as single‐copy number genes were successfully amplified from the extracted DNA samples. The DNA samples obtained by this method can be stored at ?20°C for over 1 year. Conclusions: The method was effective, easy and fast and allowed batch DNA extraction from multiple fungal isolates. Significance and Impact of Study: Use of the thermolysis method will allow researchers to obtain DNA from fungi quickly for use in molecular assays. This method requires only minute quantities of starting material and is suitable for diverse fungal species.     

A modified method for PCR-directed gene synthesis from large number of overlapping oligodeoxyribonucleotides

Here we report an improved, reproducible, simple, rapid, and cost-effective PCR-based DNA synthesis method using short (25–40 bp) overlapping oligodeoxyribonucleotides (oligos). The method involves two steps; (1) assembly of multiple/overlapping oligos by PCR to generate the template DNA and (2) amplification of the template DNA sequence with the two outermost oligos as primers. We have tested this method by synthesizing approximately 35 genes ranging in size between 300 bp and 1700 bp and G + C content from moderate (30%) to high (65%). In addition, we used the method to introduce 29 mutations simultaneously into a single gene. Key to the success of this method is the use of optimized oligo concentrations and the type of DNA polymerase used. This simplified and highly reproducible method is expected to be beneficial for the synthesis of a wide variety of genes.     

Technical note: PCR analysis of minimum target amount of ancient DNA

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low‐volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and β‐actin gene specific fragments were amplified via low‐volume PCR in a total reaction volume of 1 μl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low‐volume PCR as a promising new technique in ancient DNA analysis. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

A colorimetric confirmation method for DNA amplification in PCR and its application to the detection of Giardia lamblia cysts

A simple and quick colorimetric method for confirming DNA amplification in polymerase chain reactions (PCR) is described and has been applied to the amplification of Giardia lamblia DNA. This method detects the release of pyrophosphate based on the competition between 1,10-phenanthroline and pyrophosphate complexing with ferrous ion. When 1,10-phenanthroline complexed with Fe²⁺ is added to the finished PCR solution, depending on whether or not the DNA was amplified, the mixture is, respectively, either bleached or red. The color changed optimally for 20–30 min at 60–80 °C, and the result could be determined by detecting an absorbancy change at 510 nm or a color change discernible to the naked eye. The extent of change in absorbance was proportional to the amount of pyrophosphate produced.     

The dried corncob as a source of DNA for PCR analysis

We developed a procedure to isolate DNA from dried corncobs. This DNA was amplified successfully by PCR, producing well-defined bands in response to specific primers. The SSR patterns between cob and leaf DNA of the same inbred line were found to be identical, indicating that the DNA in the cob cells had not degraded during senescence.     

A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis

Extraction of high-quality genomic DNA fromGossypium (cotton) species is difficult due to high levels of polysaccharide, oxidizable quinones, and other interfering substances. We describe a procedure that consistently permits isolation of cotton genomic DNA of satisfactory size and quality for RFLP and PCR analysis, as well as for most routine cloning applications. Several antioxidants, phenol-binding reagents, and phenol oxidase inhibitors are used throughout the procedure, and most polysaccharides are eliminated early in the procedure by isolation of nuclei.     

Cord blood buffy coat DNA methylation is comparable to whole cord blood methylation

Cord blood DNA methylation is associated with numerous health outcomes and environmental exposures. Whole cord blood DNA reflects all nucleated blood cell types, while centrifuging whole blood separates red blood cells, generating a white blood cell buffy coat. Both sample types are used in DNA methylation studies. Cell types have unique methylation patterns and processing can impact cell distributions, which may influence comparability. We evaluated differences in cell composition and DNA methylation between cord blood buffy coat and whole cord blood samples. Cord blood DNA methylation was measured with the Infinium EPIC BeadChip (Illumina) in eight individuals, each contributing buffy coat and whole blood samples. We analyzed principal components (PC) of methylation, performed hierarchical clustering, and computed correlations of mean-centered methylation between pairs. We conducted moderated t-tests on single sites and estimated cell composition. DNA methylation PCs were associated with individual (P_PC1 = 1.4 × 10^?9; P_PC2 = 2.9 × 10^?5; P_PC3 = 3.8 × 10^-5; P_PC4 = 4.2 × 10^-6; P_PC5 = 9.9 × 10^-13, P_PC6 = 1.3 × 10^?11) and not with sample type (P_PC1-6>0.7). Samples hierarchically clustered by individual. Pearson correlations of mean-centered methylation between paired samples ranged from r = 0.66 to r = 0.87. No individual site significantly differed between buffy coat and whole cord blood when adjusting for multiple comparisons (five sites had unadjusted P<10^?5). Estimated cell type proportions did not differ by sample type (P = 0.46), and estimated proportions were highly correlated between paired samples (r = 0.99). Differences in methylation and cell composition between buffy coat and whole cord blood are much lower than inter-individual variation, demonstrating that both sample preparation types can be analytically combined and compared.