小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。     

Evaluation of a method for high yield purification of largely intact mitochondrial DNA from human placentae

We developed, and quantitatively and qualitatively evaluated an easily reproducible method for high yield purification of mitochondrial DNA (mtDNA) from human placentae by mechanical tissue disruption, differential centrifugation of mitochondria, enzymatic digestion, phenol extraction and ethanol precipitation. Average mtDNA yields were 2.5 microg/g tissue (without an RNAse treatment step) and 1.5 microg/g tissue (with an RNAse treatment step). This mtDNA migrated as a 16.5-kb isolated band in agarose gels; it yielded fragments of expected sizes after digestion with restriction enzymes; it successfully served as a template in long PCR for amplification of mtDNA sequences, and hybridized to an mtDNA probe in a predictable fashion. MtDNA yields of this method were 10-fold higher than those of previously reported ones for mtDNA purification from freshly obtained human cells and tissues, with the advantage that more placental tissue can be obtained for mtDNA purification than other types of tissue, at lower cost, and with minimal or no ethical issues.     

一种新的线粒体基因组DNA捕获探针的制备及初步应用

目的:建立新的线粒体基因组DNA杂交捕获探针制备方法并用进行初步应用。方法:通过PCR技术扩增特异线粒体DNA片段,并与生物素偶联,最后与标记磁珠的亲和素混合获得捕获探针。并自行制备的线粒体基因组DNA文库捕获探针与肝癌全基因组测序文库进行液相杂交。分离捕获产物后PCR扩增并进行测序分析。结果:成功建立了线粒体基因组杂交捕获探针制备方法并成功分离线粒体基因组DNA;对测序数据的分析显示:90%以上测序数据来自线粒体基因组DNA,且覆盖率达到100%,且均一性良好。检测到的同质性变异位点数量和异质性变异位点数量与全基因组测序数据产生的结果接近(P=0.9152,P=0.8409)。结论:新方法制备的线粒体基因组DNA杂交捕获探针可以从全基因组文库中高效捕获线粒体基因组DNA测序文库。     

Simultaneous extraction of nuclear and mitochondrial DNA from human blood

A method of simultaneous isolation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) from human blood has been proposed by improvising Lahiri's method of isolation of nuclear DNA. The approach presented here provides selectively enriched fractions and eliminates the need for two different methods or separate reagent sets for the extraction of nDNA and mtDNA. It employs an initial nuclear/ cytoplasm partitioning, followed by the similar procedural steps for the two fractions separately. It gives good quality and quantity of the nDNA as well as the mtDNA, suitable for processes like PCR amplification and sequencing and may prove to be useful for people studying population genetics and evolution using molecular markers maximizing the available resources, especially in cases where a large database needs to be generated from limited amount of blood sample. From 3 ml of blood, the yields of mtDNA salvaged from the supernatant were sufficient to set approximately 4x10(5) reactions (starting with 250 fg DNA per reactions) of mtDNA loci which otherwise would have been discarded as per original Lahiri's procedure. The quality of mtDNA from the mitochondrial fraction was suitable for all major downstream processes as confirmed by locus specific PCR amplifications and sequencing. Through this procedure, the wastage of nDNA can be avoided when mtDNA loci is studied.     

Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency.     

鱼肝线粒体DNA的简易提纯方法

本文报道一种简易分离纯化线粒体DNA(mtDNA)的方法。即以差速离心或蔗糖梯度离心制得线粒体后,用RNase除去RNA,可得纯mtDNA,或用凝胶过滤、或低熔点琼脂糖法纯化之。所获纯mtDNA可用于限制性酶切图谱的组建和其片段的克隆。此法可避开冗长的昂贵的超速离心,故可为普通实验室所采用。文中还讨论了mtDNA纯化和得率的因素。     

DNA isolation protocol for red seaweed (rhodophyta)

We report a DNA isolation protocol for red seaweed. The method is a modification of the Dellaporta et al. (1983) protocol for land plants. Our simplified version can be used to process large sample numbers and to minimise polysaccharide co-isolation. The protocol was applied to 12 red seaweed species as well as one green alga and one land plant. The protocol yields about 5 μg of high molecular weight DNA from 10 mg of dried material, with no RNA. No sign of degradation was observed after agarose gel electrophoresis for both freshly extracted DNA and DNA stored for 18 months at 4°C. DNA isolated by our protocol was suitable for genomic library construction (tested for one species), endonuclease restriction, and PCR amplification for all species.     

Isotachophoresis of nucleic acids in agarose gel rods

A new method of electrophoresis (isotachophoresis in agarose gel rods) in which nucleic acid molecules are not separated but, oppositely, are brought together into one band, was elaborated. Heterogeneous in size DNA and RNA polymers present in a few milliliters of a solution at so low concentration that their isolation by other methods is hardly attainable and fraught with losses are brought together into one visible narrow band when put in a discontinuous electric field. Polynucleotides migrate in dilute (0.1%) semifluid agarose gel that permits easy quantitative isolation of the band of interest. Resulting DNA can be used directly in PCR. The suggested method for isolation of micro amounts of nucleic acids from dilute solutions can be applied to forensic and clinical research and cancer gene diagnostics by the analysis of fragmented circulating DNA from bodily fluids.     

Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays

The extraction of genomic DNA is the crucial first step in large-scale epidemiological studies. Though there are many popular DNA isolation methods from human whole blood, only a few reports have compared their efficiencies using both end-point and real-time PCR assays. Genomic DNA was extracted from coronary artery disease patients using solution-based conventional protocols such as the phenol–chloroform/proteinase-K method and a non-phenolic non-enzymatic Rapid-Method, which were evaluated and compared vis-a-vis a commercially available silica column-based Blood DNA isolation kit. The appropriate method for efficiently extracting relatively pure DNA was assessed based on the total DNA yield, concentration, purity ratios (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀), spectral profile and agarose gel electrophoresis analysis. The quality of the isolated DNA was further analysed for PCR inhibition using a murine specific ATP1A3 qPCR assay and mtDNA/Y-chromosome ratio determination assay. The suitability of the extracted DNA for downstream applications such as end-point SNP genotyping, was tested using PCR-RFLP analysis of the AGTR1-1166A>C variant, a mirSNP having pharmacogenetic relevance in cardiovascular diseases. Compared to the traditional phenol–chloroform/proteinase-K method, our results indicated the Rapid-Method to be a more suitable protocol for genomic DNA extraction from human whole blood in terms of DNA quantity, quality, safety, processing time and cost. The Rapid-Method, which is based on a simple salting-out procedure, is not only safe and cost-effective, but also has the added advantage of being scaled up to process variable sample volumes, thus enabling it to be applied in large-scale epidemiological studies.     

Rapid and inexpensive recovery method of DNA fragments from agarose and polyacrylamide gels by a cotton-wool column tube.

We developed a rapid, convenient, simple, and inexpensive method for isolating pure DNA from agarose and polyacrylamide gels using cotton wool tubes. DNA fragments ranging in size from 193-23,130 bp can be easily recovered within 2 hours by centrifugation through cotton wool from gel slices. The recovery rate of this method is 35% to 50%, when estimated for isolation of lambda DNA-HindIII fragments. We have also recovered 700-bp polymerase chain reaction (PCR) products using cotton wool tubes from electrophoresis on both a 0.8% agarose gel and a 6% polyacrylamide gel, in which satisfactory yields of more than 50% were obtained. The DNA thus recovered in this way is biologically active and can be used as a substrate for further experimental procedures without additional purification steps.     

Affinity capture electrophoresis for sequence-specific DNA purification

A new method, affinity capture electrophoresis (ACE), has been developed for the sequence-specific isolation of DNA. The target DNA is complexed with a biotinylated probe and electrophoresed in a gel equipped with a trap of immobilized streptavidin. This selectively captures the target molecules and its biotinylated probe, while other nontarget molecules pass through the trap. The target DNA is subsequently recovered from the trap by destroying the interaction between the target DNA and the biotinylated probe. Two variations of this technique, one using triple-helix formation and the other using hybridization with a uracil-containing DNA probe at the end of the target fragment, proved effective in model experiments. Since this technique requires no denaturation and handles DNA inside an agarose gel matrix, it is, in principle, applicable to the isolation of very large DNAs.     

Noninvasive Method of DNA Isolation From Fecal Epithelial Tissue of Dairy Animals

A novel noninvasive genomic DNA isolation protocol from fecal tissue, by the proteinase K digestion and guanidine hydrochloride extraction method, was assessed for the genotyping of cattle and buffalo. The epithelial tissues present on the surface of the feces were used as source for isolation of genomic DNA. The DNA isolated from fecal tissue was found to be similar as those obtained from other body tissues such as skin, brain, liver, kidney, and muscle. The quality of DNA was checked by agarose gel electrophoresis and polymerase chain reaction (PCR). We successfully amplified a 320 bp MHC class II DRB gene and a 125 bp mt-DNA D-loop region from isolated genomic DNA of cattle. Thus, the DNA isolated using this method was suitable for common molecular biology methods, such as restriction enzyme digestion and genotyping of dairy animals through PCR.     

PCR-Free Enrichment of Mitochondrial DNA from Human Blood and Cell Lines for High Quality Next-Generation DNA Sequencing

Recent advances in sequencing technology allow for accurate detection of mitochondrial sequence variants, even those in low abundance at heteroplasmic sites. Considerable sequencing cost savings can be achieved by enriching samples for mitochondrial (relative to nuclear) DNA. Reduction in nuclear DNA (nDNA) content can also help to avoid false positive variants resulting from nuclear mitochondrial sequences (numts). We isolate intact mitochondrial organelles from both human cell lines and blood components using two separate methods: a magnetic bead binding protocol and differential centrifugation. DNA is extracted and further enriched for mitochondrial DNA (mtDNA) by an enzyme digest. Only 1 ng of the purified DNA is necessary for library preparation and next generation sequence (NGS) analysis. Enrichment methods are assessed and compared using mtDNA (versus nDNA) content as a metric, measured by using real-time quantitative PCR and NGS read analysis. Among the various strategies examined, the optimal is differential centrifugation isolation followed by exonuclease digest. This strategy yields >35% mtDNA reads in blood and cell lines, which corresponds to hundreds-fold enrichment over baseline. The strategy also avoids false variant calls that, as we show, can be induced by the long-range PCR approaches that are the current standard in enrichment procedures. This optimization procedure allows mtDNA enrichment for efficient and accurate massively parallel sequencing, enabling NGS from samples with small amounts of starting material. This will decrease costs by increasing the number of samples that may be multiplexed, ultimately facilitating efforts to better understand mitochondria-related diseases.     

食用真菌线粒体DNA的直接分析

该文报道了以限制酶HaeIII酶切13个食用菌栽培品种的总DNA,在琼脂糖凝胶上直接显现线粒体DNA带的结果。结合另外两种限制酶CfoI和MSpI的酶切结果和HaeIII在其它真菌中的研究报道,提出了利用识别四个GC对的限制酶酶切真菌总DNA,直接分析线粒体DNA的方法。     

Quantification of mitochondrial DNA copy number: Pre-analytical factors

Mitochondrial DNA (mtDNA) content is important for understanding many cellular processes. Several pre-analytical factors, from sample collection to DNA extraction can affect measurement of mtDNA copy number. In the present study, whole blood samples yielded a higher mtDNA copy number than buffy coat samples. mtDNA content is affected by the cell separation method used and the time between blood withdrawal and cell separation. Thus, reference values must be established with the same type of sample. As to the DNA isolation and purification method, the manual phenol method can give randomly false high values. The QIAamp DNA Mini Kit provided the most highly reproducible mtDNA/nDNA yield.     

Detection of Helicobacter pylori DNA in human feces by PCR: DNA stability and removal of inhibitors

In this study, the stability of Helicobacter pylori DNA in human feces and the effect of a diet lacking in plant material, the suspected source of PCR inhibitors in human feces, were investigated. In addition, a method to remove these inhibitors was developed. Stools inoculated with H. pylori were used as a model. For this purpose, a H. pylori suspension (10(8) CFU/ml) was used to spike stool samples obtained from four healthy adults known to be H. pylori negative. The evaluation of the stability of H. pylori DNA in feces showed that DNA was degraded after 3 days of contact with fecal material at 37 degrees C. A 2-day diet completely free of plant material was sufficient to eliminate PCR inhibitors from human feces. However, inhibitors were detected 48 h after a normal diet was resumed. A new technique consisting of agarose blocks containing embedded DNA as a template for PCR amplification was used for removal of inhibitors, following DNA extraction by a modified QIAamp tissue method (Qiagen, Hilden, Germany). When this method was applied to inhibiting stool samples known to have an inhibitory effect and spiked with H. pylori (5.10(8) CFU/g), a positive PCR was obtained showing that inhibitors present in the original DNA samples were completely removed. The agarose embedded DNA block method is highly efficient and provides clean, high quality template DNA for PCR purposes avoiding long and fastidious conventional extraction methods. In conclusion, this study confirms that H. pylori DNA degrades with time in stools. A diet free of plant material or a special DNA preparation can be used to remove inhibitors and to allow the detection of H. pylori.     

Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping.

A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.     

DNA microarray probe preparation by gel isolation nested PCR

To develop a simplified method that can rapidly prepare DNA microarray probes in a massive scale, a lambda phage genomic DNA-fragments library was constructed for the microarray-probes collection. Four methods of DNA band recovery from the first PCR products were tested and compared. The DNA microarray probes were collected by a novel method of nested PCR that was mediated by gel isolation of the first PCR products. This method was named GIN-PCR. The probes that were prepared by this GIN-PCR technique were used as subjects to fabricate a DNA microarray. The results showed that a wooden toothpick was superior to the other 3 methods, since this technique can steadily transfer the DNA bands as the template of the second PCR after the first PCR. A group of probes were successfully collected and DNA microarrays were constructed using these probes. Hybridization results demonstrated that this technique of DNA recovery and probe preparation was rapid, efficient, and effective. We developed a cost-effective and less labor-intensive method for DNA microarray probe preparation by nested PCR that is mediated by wooden toothpick transfer of the DNA bands in the gel after electrophoresis.     

DNA isolation from forest soil suitable for PCR assays of fungal and plant rRNA genes

This protocol for DNA isolation from forest soil samples is advantageous because it uses only one liquid transference step and can process several samples with minimal time and equipment. The use of benzyl chloride early in the extraction protocol increases DNA yield and purity. The obtained DNA is useful for PCR amplification of nuclear and mitochondrial ribosomal related sequences from fungi and ribosomal DNA from plant chloroplasts. Isolated DNA can be used either undiluted or at low dilutions in PCR assays. A final glassmilk treatment of isolated DNA is useful to recover high molecular weight DNA fractions from agarose gel. DNA losses during glassmilk treatment can generate negative PCR results.     

An improved method of plant megabase DNA isolation in agarose microbeads suitable for physical mapping and YAC cloning

The isolation of high quality megabase DNA from plant cells that is susceptible to a variety of molecular reagents is a critical first step in the physical analysis of complex genomes. A method for the isolation of such DNA by encapsulating plant protoplasts in agarose microbeads is presented. In comparison with the conventional agarose plug method, microbeads provide a dramatic increase in the surface area yielding megabase DNA that can be treated essentially as an aqueous DNA solution. Examples of the utility of DNA prepared by this technique for physical mapping, partial restriction enzyme digestion and cloning of large inserts as YACs are presented.