单细胞凝胶电泳技术操作方法介绍

单细胞凝胶电泳技术是一种测定和研究单个细胞DNA链断裂的新电泳技术。具有简便、快速、灵敏、样品量少、无需放射性标记等特点。介绍其实验方法并就细胞悬液和胶板的制备,裂解、解旋和电泳,中和和染色,图像分析等操作步骤和方法予以介绍。     

用单细胞凝胶电泳技术检测粉纹夜蛾5BI细胞DNA损伤的研究

为了探测昆虫细胞的DNA损伤,本实验应用单细胞凝胶电泳技术,对不同剂量的过氧化氢和甲醛引起的粉纹夜蛾5BI细胞DNA损伤效应进行了研究,结果表明59μM=10μM、150μM的过氧化氢能引起粉纹夜蛾5BI细胞的DNA断裂,且断裂程度和浓度水平之间成正相关关系;10μM、30μM、50μM的甲醛能引起粉纹夜蛾5BI细胞DNA损伤,其中在10μM时引起DNA断裂,在30μM、50μM时引起DNA交联。     

单细胞凝胶电泳应用研究进展

单细胞凝胶电泳技术是被广泛使用的检测DNA损伤的技术,其具有灵敏度高、简便、经济、快速等各种优点。在近30年来,该技术广泛被应用于遗传毒理、环境监测、医学诊断等研究及各类实验证实方面。近些年单细胞凝胶电泳与荧光原位杂交等其他技术相结合后更是拓宽了它的应用范围,本文主要介绍了单细胞凝胶电泳的原理及优劣,并重点阐明了其在遗传毒理学、细胞凋亡、环境监测、群体生物学、临床诊断与治疗,生物信息学领域上的应用,并讨论了它的发展前景。     

The study of DNA integrity in somatic cells and spermatozoa by single cell gel electrophoresis with silver staining

In present work we studied DNA damage in human and bovine lymphocytes and spermatozoa by means of single cell gel electrophoresis followed by silver staining. The spontaneous frequency of DNA damage estimated manually in spermatozoa from healthy donors did not exceed 9% (on average -- 4.8 +/- 1.2%). The frequency of DNA damages in bull sperm after short (less than a year) and long period (more than 20 years) of cryopreservation was assessed as 3.1 +/- 0.9 and 4.3 +/- 0.5%, correspondingly. The comparative estimation of DNA damages in lymphocytes followed by silver staining is a valuable tool to estimate DNA damage in populations of somatic and reproductive cells.     

High resolution mini-two-dimensional gel electrophoresis of yeast ribosomal proteins

By applying two-dimensional gel electrophoresis (basic urea/acidic urea) in a mini-form yeast ribosomal proteins were separated with a high resolution. On basis of this separation pattern a standard nomenclature for the yeast ribosomal proteins is proposed.     

Genotoxicity of vanadium pentoxide evaluate by the single cell gel electrophoresis assay in human lymphocytes

Vanadium compounds are extensively used in modern industry and occupational exposure to high doses of Vanadium is quite common. In this study, the genotoxicity of vanadium pentoxide (V₂O₅) was evaluated directly in whole blood leukocytes and in human lymphocyte cultures using the single-cell gel electrophoresis assay (Comet Assay) to detect DNA damage expressed as DNA strand breaks and alkali labile sites. This chemical produces a clear dose-response in DNA migration in whole blood leukocytes and a significative positive effect only with the highest tested concentration in human lymphocyte cultures. After different recovery times the level of DNA damage returned to the control values. These results indicate that V₂O₅ is capable to induce DNA single-strand breaks and/or alkali-labile damage.     

Application and evaluation of denaturing gradient gel electrophoresis to analyse the yeast ecology of wine grapes

The performance of denaturing gradient gel electrophoresis (DGGE) for analysing yeasts associated with wine grapes was compared with cultural isolation on malt extract agar (MEA). After optimisation of PCR and electrophoretic conditions, the lower limit of yeast detection by PCR-DGGE was 10(2) cfuml(-1), although this value was affected by culture age and the relative populations of the species in mixed culture. In mixed yeast populations, PCR-DGGE detected species present at 10-100-fold less than other species but not when the ratio exceeded 100-fold. Aureobasidium pullulans was the main species isolated from immature, mature, and both damaged and undamaged grapes. It was not detected by PCR-DGGE when present at populations less than 10(3) cfug(-1). When approaching maturity, damaged grapes gave a predominance of Metschnikowia and Hanseniaspora species (10(5)-10(7) cfug(-1)), all detectable using PCR-DGGE. However, various species of Rhodotorula, Rhodosporidium and Cryptococcus were not detected by this method, even when populations were as high as 10(4) cfug(-1). PCR -DGGE was less sensitive than culture on MEA for determining the yeast ecology of grapes and could not reliably detect species present at populations less than 10(4) cfug(-1). However, this method detected a greater diversity of species than agar plating.     

Evaluation of benzo(a)pyrene-induced DNA damage in human endothelial cells using alkaline single cell gel electrophoresis

The alkaline version of the 'comet assay' was used to evaluate DNA damage in human umbilical vein endothelial cells (HUVEC) exposed to 0.1, 1.0, or 10 microM benzo(a)pyrene for 90min. The genotoxicity was monitored in HUVEC pretreated with the Ah-receptor agonist beta-naphthoflavone (BNF), previously shown to induce cytochrome P4501A1 (CYP1A1) activity in these cells, and in vehicle-treated HUVEC with only constitutive levels of this enzyme. Increased DNA damage was observed only in cells that had been exposed to 10 microM benzo(a)pyrene, cells exposed to BNF being subjected to the most extensive damage. The CYP1A/B-inhibitor alpha-naphthoflavone (ANF) reduced the benzo(a)pyrene-induced DNA-damage in the BNF-treated HUVEC to the same level as in the uninduced cells. The fact that benzo(a)pyrene induced DNA damage in vehicle-treated HUVEC suggests that there may be at least one alternative route of bioactivation for benzo(a)pyrene in these cells. Consequently, judging from the present results it seems as if tobacco-related polycyclic aromatic hydrocarbons (PAHs) may disrupt the function of the endothelial lining in blood vessels with low monooxygenase activity. It is proposed that exposure to Ah receptor agonists via, for example, tobacco smoke, may enhance the DNA-damaging effects of smoke-related genotoxic PAHs in human endothelial cells. The role of PAHs in endothelial dysfunction of tobacco smokers should therefore be further studied.     

Study on proteins from yeast cytoplasmic ribosomes by two-dimensional gel electrophoresis

Summary Proteins of yeast cytoplasmic ribosomes were analyzed by two different methods of two-dimensional gel electrophoresis: run at pH 8.6 in 1-D¹ and at pH 4.6 in 2-D (Method A); run at pH 5.0 in 1-D and in the presence of sodium dodecyl sulfate in 2-D (Method B). The numbers of proteins estimated were 28 (Method A) and 29 or 30 (Method B) in the 40S small subunit, and 40 (Method A) and 41 (Method B) in the 60S large subunit, respectively. Molecular weights of proteins in the small and the large subunits were found to be less than 40,000 and 60,000 respectively.     

Rearrangements of yeast chromosomes revealed by pulsed field gel electrophoresis

Electrophoretic karyotypes of yeast Saccharomyces cerevisiae integrant strains containing the pYF91 plasmid integrated into the chromosomes I, III, VI, IX, XI were studied. A possibility was demonstrated of visual identification of the chimaeric chromosomes via the molecular weight increase by 13200 bp (the plasmid size) determined by pulsed field gel electrophoresis. Several gamma-rays induced rearrangements of the yeast chimaeric chromosome I causing instability in hybrids were also studied. The deletions induced in the I chromosome were analysed and their size estimated. The technique of pulsed field gel electrophoresis is recommended for determination of insertions and deletions in the chromosomes of yeast Saccharomyces cerevisiae.     

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.     

Evaluation of the effect of 90Sr beta-radiation on human blood cells by chromosome aberration and single cell gel electrophoresis (comet assay) analysis

Among various environmental genotoxins, ionizing radiation has received special attention because of its mutagenic, carcinogenic and teratogenic potential. In this context and considering the scarcity of literature data, the objective of the present study was to evaluate the effect of 90Sr beta-radiation on human cells. Blood cells from five healthy donors were irradiated in vitro with doses of 0.2-5.0Gy from a 90Sr source (0.2Gy/min) and processed for chromosome aberration analysis and for comet assay. The cytogenetic results showed that the most frequently found aberration types were acentric fragments, double minutes and dicentrics. The alpha and beta coefficients of the linear-quadratic model, that best fitted the data obtained, showed that 90Sr beta-radiation was less efficient in inducing chromosome aberrations than other types of low linear energy transfer (LET) radiation such as 3H beta-particles, 60Co gamma-rays, 137Cs and 192Ir and X-rays. Apparently, 90Sr beta-radiation in the dose range investigated had no effect on the modal chromosome number of irradiated cells or on cell cycle kinetics. Concerning the comet assay, there was an increase in DNA migration as a function of radiation dose as evaluated by an image analysis system (tail moment) or by visual classification (DNA damage). The dose-response relation adequately fitted the non-linear regression model. In contrast to the cytogenetic data, 90Sr beta-radiation induced more DNA damage than 60Co gamma-radiation when the material was analyzed immediately after exposures. A possible influence of selective death of cells damaged by radiation was suggested.     

Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis

A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20-50 sec in our experiments, can be adjusted to optimize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of self-ligated lambda DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed.     

Separation of yeast chromosome-sized DNAs by pulsed field gradient gel electrophoresis

A new type of gel electrophoresis separates DNA molecules up to 2000 kb with resolutions exceeding the logarithmic molecular weight dependence of conventional electrophoresis. The technique uses 1.5% agarose, 10 to 20 micrograms of DNA per well, and low ionic strength buffers. It employs alternately pulsed, perpendicularly oriented electrical fields, at least one of which is inhomogeneous. The duration of the applied electrical pulses is varied from 1 sec to 90 sec to achieve optimal separations for DNAs with sizes from 30 to 2000 kb. This pulsed field gradient gel electrophoresis fractionates intact S. cerevisiae chromosomal DNA, producing a molecular karyotype that greatly facilitates the assignment of genes to yeast chromosomes. Each yeast chromosome consists of a single piece of DNA; the chromosome sizes are consistent with the genetic linkage map. We also describe a general method for preparing spheroplasts, and cell lysates, without significant chromosomal DNA breakage.     

Detection of single nucleotide polymorphisms by PCR conformation-difference gel electrophoresis

The most common genetic variations in the human genome, single nucleotide polymorphisms (SNPs), are ideal biomarkers and are used extensively in disease research. Here we introduce a novel method of PCR-conformation-difference gel electrophoresis (PCR-CDGE) used for detecting SNPs. The principle of PCR-CDGE relies PCR products from different homozygous DNA samples showing dissimilar migration patterns upon PAGE due to their conformational differences. PCR products from heterozygous DNA samples may exhibit two or more bands in PAGE because of the existence of DNA homoduplexes and heteroduplexes. In this study, analysis of two SNPs showed that PCR-CDGE is an accurate, simple, rapid, low-cost, and high-throughput genotyping method that could be used in most laboratories.     

Alkaline single cell gel electrophoresis and human biomonitoring for genotoxicity: a study on subjects with residential exposure to radon.

Based on theoretical estimates and various correlation studies, it has been suggested that ingestion of radon in drinking water represents an increased risk for cancer. Such a risk has never been conclusively shown in epidemiological or experimental animal studies, however, and it has been questioned whether the radon level in the drinking water is of any significance in terms of overall radon exposure. Using primary DNA damage as a biological marker for an ongoing exposure to ionising radiation, the present study was undertaken to investigate whether people with different types of residential radon exposures differed with regard to their levels of DNA damage in circulating lymphocytes. DNA damage was measured in coded blood samples from 125 residents living in 45 households with different levels of radon-222 in the drinking water (10-2410 Bq/l) and indoor air (35-1025 Bq/m3) using alkaline single cell gel electrophoresis (the 'Comet' assay). Increased levels of radon in indoor air (>200 Bq/m3) were found to be associated with an increased level of DNA damage in peripheral lymphocytes (P相似文献