A rapid inexpensive method for the isolation of restrictable mitochondrial DNA from various plant sources

A simplified method for the isolation of mitochondrial DNA (mtDNA) of several plant species from either coleoptile or tissue cultured cells is described. The procedure does not require gradient ultracentrifugation or organic solvent extractions (such as phenol, chloroform, ether, etc.). Protoplast isolation is not required for the release of organelles from cell suspension cultured cells. The entire procedure can be performed in a single day and employs differential low speed centrifugations for isolation of mitochondria and differential precipitations for the recovery of restrictable DNA.     

Secondary structure of nascent DNA of Ehrlich ascites tumor cells obtained by nitrocellulose column chromatography

Pulse labeled DNA was isolated from EHRLICH ascites cells using different methods. Depending on the isolation procedure, the nascent DNA separated from the bulk DNA by nitrocellulose column chromatography was either entirely double stranded or contained single stranded constituents. This seems to be due to the destabilized state of the nascent DNA within a living cell causing the partial conversion of newly replicated DNA to the single stranded form when certain DNA isolation methods are applied. It is suggested that the nascent DNA separated by nitrocellulose chromatography is normally double stranded.     

应用光学微操作技术分选单条水稻染色体

报道了一种基于光镊技术的实用的单条染色体分选技术。具体介绍了用光镊与光刀结合、并辅以微吸管分选水稻单条染色体的过程。通过该方法得到的水稻单条染色体样品经过分子克隆,制备出了染色体特异的DNA片段并用于水稻基因组测序工作。还将光学微操作技术与现有的几种分选单条染色体的方法(如玻璃微针挑取、激光弹射以及流式细胞仪等)进行了比较。与这些方法相比,光学微操作方法具有液相环境中分离、操作简易、对染色体损伤小、选择性高、无污染等优点。     

激光显微切割分选少量胃癌细胞中PSCA基因的SNP分析

随着基因组关联分析方法的应用,越来越多与胃癌相关的易感基因被发现．易感基因的多态性检测已逐步进入胃癌临床诊断和研究．然而,利用少量胃粘膜细胞开展单核苷酸多态性（SNP）分析对胃癌进行早期诊断常遇下述困难,一是少量胃癌细胞混杂在多种细胞中,异常信号常易被淹没,二是细胞量极少,因此获得的基因组DNA量微,进行多位点或全基因组分析存在困难. 本文利用激光显微切割技术分选少量胃癌细胞,结合全基因组放大技术,进行胃癌相关的前列腺干细胞抗原基因(PSCA)的SNP分析．通过聚合酶链反应-限制性片段长度多态性(PCR-RFLP)和克隆测序方法分析,在分选的胃癌细胞中检测到PSCA的rs2976392位点胃癌相关的“A”等位与rs2294008位点胃癌相关的“T”等位．研究结果表明,所采用的全基因组放大方法保真性高,经过分选的胃癌细胞中SNP位点的检测灵敏度和可靠性大为提高．所建立的少量细胞基因多位点检测方法将同样应用于其它肿瘤和组织的少量细胞研究中,全基因组放大产物也可进行高通量的基因芯片和第二代测序研究．     

Simultaneous isolation of high molecular weight RNA and DNA from limited amounts of tissues and cells

A simple method is described for the simultaneous isolation of both DNA and RNA from tissues and cultured cells obtainable in limited quantities only. The method is based on a suitable combination of steps designed for preparations of high molecular weight nucleic acids in cases when restricted amounts of tissues like small-sized and unique biopsies of tumors are available for studies of gene organization and expression. Using this protocol, undegraded total RNA suitable for Northern blot analysis and high molecular weight DNA for Southern blots was obtained from various sources (mammary and colon carcinomas, meningiomas, colonic and placental tissue, and several cell cultures).     

Polymerase chain reaction in diagnosis of toxoplasmosis of the central nervous system: effect of methods for isolating DNA from samples of cerebrospinal fluid on results of the reaction

We examined influence of the method of isolation of DNA from cerebrospinal fluid samples on results of PCR in the diagnosis of toxoplasmosis of the central nervous system. Three different protocols of DNA isolation were used for DNA extraction from 360 samples made of cerebrospinal fluid spiked with tachyzoites of Toxoplasma gondii: thermic, enzymatic and enzymatic-filtering. Purified DNA samples were tested by PCR with primers T15 and T16 designed for the B1 gene of the parasite. Enzymatic method of DNA isolation appeared most effective allowing detection of T. gondii DNA in 50% of samples containing single parasite cell.     

Isolation of Plasmid DNA rescued from single colonies of<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> by means of rolling circle amplification

We report a simple method to isolate plasmids from single colonies ofAgrobacterium tumefaciens by means of rolling circle amplification. The amplified DNA can be digested by restriction enzymes for plasmid verification and transformed intoEscherichia coli for plasmid rescue. Compared with conventional procedures, this method eliminates liquid culturing ofAgrobacterium cells and subsequent DNA isolation and enables large-scale plasmid analyses.     

Automatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non‐invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low‐volume on‐chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non‐related individuals. Single‐cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non‐related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single‐cell basis.     

A simple and efficient method for isolating trichomes for downstream analyses

Arabidopsis trichomes are an excellent cell type to address many questions in plant biology including the control of cell shape, endoreplication, and cell expansion. Because trichomes comprise such a small percentage of the cells of a leaf, biochemical analyses of trichomes are limited. To overcome this limitation, we developed a method for removing trichomes from the leaf surface. Our method allows the isolation of intact trichomes for use in downstream applications such as cell wall analysis, immunolocalization of trichome proteins, analysis of DNA content, and proteomics. Also, this method will facilitate the isolation of trichomes from practically any plant species.     

DNA isolation from fresh,dry plant samples with highly acidic tissue extracts

Current DNA isolation methods are limited in their ability to obtain quality and/or quantity DNA from plants, such asEmblica officinalis, Terminalia belerica, andTerminalia chebula, which have low pH and high amounts of secondary metabolites in tissue extracts. Our modified DNA isolation method yields good-quality, high-molecular-weight DNA that is free of contaminants and colored pigments and is suitable for PCR amplification. This method is also useful for isolating DNA from dry powders.     

Characterization of a novel microfluidic platform for the isolation of rare single cells to enable CTC analysis from head and neck squamous cell carcinoma patients

Detailed examination of tumor components is leading‐edge to establish personalized cancer therapy. Accompanying research on cell‐free DNA, the cell count of circulating tumor cells (CTCs) in patient blood is seen as a crucial prognostic factor. The potential of CTC analysis is further not limited to the determination of the overall survival rate but sheds light on understanding inter‐ and intratumoral heterogeneity. In this regard, commercial CTC isolation devices combining an efficient enrichment of rare cells with a droplet deposition of single cells for downstream analysis are highly appreciated. The Liquid biopsy platform CTCelect was developed to realize a fully‐automated enrichment and single cell dispensing of CTCs from whole blood without pre‐processing. We characterized each process step with two different carcinoma cell lines demonstrating up to 87 % enrichment (n = 10) with EpCAM coupled immunomagnetic beads, 73 % optical detection and dispensing efficiency (n = 5). 40 to 56.7 % of cells were recovered after complete isolation from 7.5 ml untreated whole blood (n = 6). In this study, CTCelect enabled automated dispensing of single circulating tumor cells from HNSCC patient samples, qPCR‐based confirmation of tumor‐related biomarkers and immunostaining. Finally, the platform was compared to commercial CTC isolation technologies to highlight advantages and limitations of CTCelect. This system offers new possibilities for single cell screening in cancer diagnostics, individual therapy approaches and real‐time monitoring.     

Large-scale purification of plasmid DNA by anion-exchange high-performance liquid chromatography.

Numerous methods have previously been reported for the final steps in the large-scale purification of plasmid DNA. Although gel permeation and reverse-phase high-performance liquid chromatography have been utilized for this procedure in the past, the limited capacity of these systems often necessitated multiple rounds of chromatography, especially with the high copy number plasmids commonly in use today. In this paper, the use of the high-capacity, high-resolution Protein-Pak DEAE 8HR column is presented for the large-scale isolation of highly purified plasmid DNA from crude E. coli cell lysates. Up to 5 mg of plasmid DNA have been purified in a single 50-minute chromatography run. The purified DNA demonstrated excellent biological activity as demonstrated by restriction endonuclease digestion, E. coli transformation and DNA-mediated gene transfection of eukaryotic cells.     

Genomic sequencing of single microbial cells from environmental samples

Recently developed techniques allow genomic DNA sequencing from single microbial cells [Lasken RS: Single-cell genomic sequencing using multiple displacement amplification. Curr Opin Microbiol 2007, 10:510-516]. Here, we focus on research strategies for putting these methods into practice in the laboratory setting. An immediate consequence of single-cell sequencing is that it provides an alternative to culturing organisms as a prerequisite for genomic sequencing. The microgram amounts of DNA required as template are amplified from a single bacterium by a method called multiple displacement amplification (MDA) avoiding the need to grow cells. The ability to sequence DNA from individual cells will likely have an immense impact on microbiology considering the vast numbers of novel organisms, which have been inaccessible unless culture-independent methods could be used. However, special approaches have been necessary to work with amplified DNA. MDA may not recover the entire genome from the single copy present in most bacteria. Also, some sequence rearrangements can occur during the DNA amplification reaction. Over the past two years many research groups have begun to use MDA, and some practical approaches to single-cell sequencing have been developed. We review the consensus that is emerging on optimum methods, reliability of amplified template, and the proper interpretation of 'composite' genomes which result from the necessity of combining data from several single-cell MDA reactions in order to complete the assembly. Preferred laboratory methods are considered on the basis of experience at several large sequencing centers where >70% of genomes are now often recovered from single cells. Methods are reviewed for preparation of bacterial fractions from environmental samples, single-cell isolation, DNA amplification by MDA, and DNA sequencing.     

An assessment of the efficiency of fungal DNA extraction methods for maximizing the detection of medically important fungi using PCR

To date, no single reported DNA extraction method is suitable for the efficient extraction of DNA from all fungal species. The efficiency of extraction is of particular importance in PCR-based medical diagnostic applications where the quantity of fungus in a tissue biopsy may be limited. We subjected 16 medically relevant fungi to physical, chemical and enzymatic cell wall disruption methods which constitutes the first step in extracting DNA. Examination by light microscopy showed that grinding with mortar and pestle was the most efficient means of disrupting the rigid fungal cell walls of hyphae and conidia. We then trialled several published DNA isolation protocols to ascertain the most efficient method of extraction. Optimal extraction was achieved by incorporating a lyticase and proteinase K enzymatic digestion step and adapting a DNA extraction procedure from a commercial kit (MO BIO) to generate high yields of high quality DNA from all 16 species. DNA quality was confirmed by the successful PCR amplification of the conserved region of the fungal 18S small-subunit rRNA multicopy gene.     

A general method for isolation of high molecular weight DNA from eukaryotes.

A new method for isolation of high molecular weight DNA from eukaryotes is presented. This procedure allows preparation of DNA from a variety of tissues such as calf thymus or human placenta and from cells which were more difficult to lyse until now (e.g. Crypthecodinium cuhnii, a dinoflagellate). The DNA obtained in such a way has an average molecular weight of about 200 X 10(6) d and contains very few, if any, single strand breaks.     

Protein-DNA crosslinking in reconstituted nucleohistone, nuclei and whole cells by picosecond UV laser irradiation.

A picosecond UV laser was used to cross-link proteins to DNA in nuclei, whole cells and reconstituted nucleohistone. Irradiation of the nucleohistone resulted in crosslinking 15-20% of bound histones to DNA in a very short time (one or several picosecond pulses), the efficiency of crosslinking to single stranded DNA being higher than to double stranded DNA. All histones as well as high mobility group 1 proteins were identified in the covalently linked protein-DNA complexes upon irradiation of isolated nuclei and whole cells. A method is suggested for isolation of crosslinked material from cells and nuclei in amounts sufficient for further analysis. Experiments with reconstituted nucleohistones showed that upon irradiation at a constant dose the efficiency of crosslinking depended on the intensity of the light, thus suggesting a two-quantum process is involved in the reaction.     

Simultaneous purification of chloroplast and mitochondrial DNA without isolation of intact organelles from green carrot tissue and suspension cultures

We report here the simultaneous purification of chloroplast (cpDNA) and mitochondrial DNA (mtDNA) from green tissue and suspension cultures of carrot without organelle isolation. This method is based on isolating total nucleic acids from frozen tissue and separating the nuclear, chloroplast and mitochondrial fractions using sequential isopycnic sedimentation in two gradients of cesium chloride containing bisbenzamide. From 10 g of mature carrot leaves, 10 to 30μg of organelle DNA was consistently recovered from mature carrot leaves, while 30 to 50 μg was recovered from suspension cells. The method can be used to isolate chloroplast and mitochondrial DNAs from single plants without sacrificing the individual.     

Impact of the comet assay in radiobiology

Until the development of single cell gel electrophoresis methods in the 1980s, measurement of radiation-induced DNA strand breaks in individual cells was limited to detection of micronuclei or chromosome breaks that measured the combined effects of exposure and repair. Development of methods to measure the extent of migration of DNA from single cells permitted detection of initial radiation-induced DNA breaks present in each cell. As cells need not be radiolabeled, there were new opportunities for analysis of radiation effects on cells from virtually any tissue, provided a single cell suspension could be prepared. The comet assay (as this method was subsequently named) was able to measure, for the first time, the fraction of radiobiologically hypoxic cells in mouse and human tumors. It was used to determine that the rate of rejoining of DNA breaks was relatively homogenous within an irradiated population of cells. Because individual cells were analyzed, heavily damaged or apoptotic cells could be identified and eliminated from analysis to determine "true" DNA strand break rejoining rates. Other examples of applications of the comet assay in radiobiology research include analysis of the inter-individual differences in response to radiation, effect of hypoxia modifying agents on tumor hypoxic fraction, the role of cell cycle position during DNA break induction and rejoining, non-targeted effects on bystander cells, and effects of charged particles on DNA fragmentation patterns.