Trapping and breaking of in vivo nicked DNA during pulsed field gel electrophoresis

Pulsed field gel electrophoresis (PFGE) offers a high-resolution approach to quantify chromosomal fragmentation in bacteria, measured as percentage of chromosomal DNA entering the gel. The degree of separation in pulsed field gel (PFG) depends on the size of DNA as well as various conditions of electrophoresis such as electric field strength, time of electrophoresis, switch time, and buffer composition. Here we describe a new parameter, the structural integrity of the sample DNA itself, that influences its migration through PFGs. We show that subchromosomal fragments containing both spontaneous and DNA damage-induced nicks are prone to breakage during PFGE. Such breakage at single-strand interruptions results in artifactual decrease in molecular weight of linear DNA making accurate determination of the number of double-strand breaks difficult. Although breakage of nicked subchromosomal fragments is field strength independent, some high-molecular-weight subchromosomal fragments are also trapped within wells under the standard PFGE conditions. This trapping can be minimized by lowering the field strength and increasing the time of electrophoresis. We discuss how breakage of nicked DNA may be mechanistically linked to trapping. Our results suggest how to optimize conditions for PFGE when quantifying chromosomal fragmentation induced by DNA damage.     

脉冲场凝胶电泳技术及其在真菌学研究中的应用

脉冲电泳是用于分离大分子量DNA的一种电泳技术,已广泛用于真菌的核型分析,种群特异性鉴定,基因定位及遗传分析的研究。介绍了脉冲电泳的原理,发展和基本操作程序,并阐述了脉冲电泳技术在真菌分子生物学研究领域的应用。     

Assignment of Alu-repetitive sequences to large restriction fragments from human chromosomes 6 and 22

We have employed a pulsed field gel electrophoresis and Alu hybridization approach for identification of large restriction fragments on chromosome 6 and 22. This technique allows large portions of selected human chromosomes to be visualized as discrete hybridization signals. Somatic cell hybrid DNA which contains chromosome 6 or chromosome 22 was restricted with either Notl or Mlul. The restriction fragments were separated by pulsed field gel electrophoresis (PFGE) and hybridized against an Alu repetitive sequence (Blur 8). The hybridization signals result in a fingerprint-like pattern which is unique for each chromosome and each restriction enzyme. In addition, a continuous pattern of restriction fragments was demonstrated by gradually increasing puls times. This approach will also be suitable to analyze aberrant human chromosomes retained in somatic cell hybrids and can be used to analyze flow sorted human chromosomes. To this end, our method provides a valuable alternative to standard cytogenetic analysis.     

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.     

Shotgun法测序制备高纯度水稻基因组DNA的方法探讨(英)

在用散弹 (shotgun)法测定水稻 (OryzasativaL .ssp .indica)基因组全序列的过程中 ,叶绿体和线粒体DNA的污染问题非常严峻 .应用脉冲场电泳 (PFGE)技术对水稻基因组DNA进行纯化 ,结果表明它能够有效去除叶绿体和线粒体DNA ,使其污染率从 3%降低到 0 2 % .同时 ,比较了水稻绿苗和黄化苗的DNA得率 ,以及HB法和NIB法制备大分子质量(HMW)DNA的异同 .最后提出一套制备水稻基因组DNA的方法 ,包括黄化苗培养 ;细胞核的分离、包埋和裂解 ;脉冲场电泳纯化、回收聚集在低熔点 (LMP)胶中的水稻HMWDNA .用该方法所得的水稻基因组DNA ,纯度高 (无叶绿体和线粒体DNA污染 )、基因组完整 (机械剪切和降解少 )、回收率高 (操作过程中DNA损失少 ) .另外 ,首次报道在融化的低熔点(LMP)胶中对水稻HMWDNA于 38℃进行超声波处理 ,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1 5～ 3kb ,3～ 12kb) ,并且效果优于在TE中进行操作     

A small (58-nm) attached sphere perturbs the sieving of 40-80-kilobase DNA in 0.2-2.5% agarose gels: analysis of bacteriophage T7 capsid-DNA complexes by use of pulsed field electrophoresis.

Although the icosahedral bacteriophage T7 capsid has a diameter (58 nm) that is 234-fold smaller than the length of the linear, double-stranded T7 DNA, binding of a T7 capsid to T7 DNA is found here to have dramatic effects on the migration of the DNA during both pulsed field agarose gel electrophoresis (PFGE; the field inversion mode is used) and constant field agarose gel electrophoresis (CFGE). For these studies, capsid-DNA complexes were obtained by expelling DNA from mature bacteriophage T7; this procedure yields DNA with capsids bound at a variable position on the DNA. When subjected to CFGE at 2-6 V/cm in 0.20-2.5% agarose gels, capsid-DNA complexes arrest at the electrophoretic origin. Progressively lowering the electrical potential gradient to 0.5 V/cm results in migration; most complexes form a single band. The elevated electrical potential gradient (3 V/cm) induced arrest of capsid-DNA complexes is reversed when PFGE is used instead of CFGE. For some conditions of PFGE, the mobility of capsid-DNA complexes is a function of the position of the capsid on the DNA. During either CFGE (0.5 V/cm) or PFGE, capsid-DNA complexes increasingly separate from capsid-free DNA as the percentage of agarose increases. During these studies, capsid-DNA complexes are identified by electron microscopy of enzymatically-digested pieces of agarose gel; this is apparently the first successful electron microscopy of DNA from an agarose gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulsed field gel electrophoresis of chromosomal DNA reveals a clonal population structure to Bacillus thuringiensis that relates in general to crystal protein gene content

Seventy strains of Bacillus thuringiensis representing 21 serovars were allocated to 38 genomic groups using pulsed field gel electrophoresis (PFGE) of restriction enzyme-digested DNA. There was a broad correlation between PFGE type and serotype for serovars darmstadiensis, israelensis, kenyae, kumamotoensis, kurstaki, sotto, thuringiensis, and tolworthi, although some serovars included atypical strains. Serovars canadensis and entomocidus were heterogeneous. Detection of crystal protein genes by polymerase chain reaction indicated an approximate correlation between PFGE type and cry gene complement. For example, cry1 products were amplified from DNA from PFGE type 17 strains of serovar aizawai and from PFGE type 23 strains of serovar tolworthi but not from a PFGE 18 strain of aizawai nor from a PFGE type 24 strain of tolworthi. These data suggest a clonal population structure to B. thuringiensis with some consistency of Cry-plasmid composition within PFGE types.     

A method for two-dimensional DNA electrophoresis (2D-DE): application to the immunoglobulin heavy chain variable region

The scarcity of single-copy probes creates difficulty in the generation of large-scale physical maps of mammalian gene families. A simple method of two-dimensional DNA electrophoresis (2D-DE) has been developed to overcome this problem. DNA (2 micrograms) is digested with a rare-cutting restriction endonuclease and size separated by pulsed-field gel electrophoresis (PFGE). The DNA, still contained within the lane of the PFGE gel, is digested with a second frequent-cutting restriction enzyme and is subjected to an electrical field perpendicular to that of the PFGE. 2D-DE allows the simultaneous mapping, to large restriction fragments, of all the genes detected by a particular probe. The human immunoglobulin variable region was used as an example for this procedure. Two VH5 genes, on 8- and 9-kb EcoRI fragments, were mapped to 200- and 65-kb SfiI fragments, respectively, by 2D-DE. This technique will be particularly useful in the generation of physical maps of complex human gene families and of repeat families.     

Moving pictures of DNA released upon lysis from bacteria,chloroplasts, and mitochondria

Summary Cells and organelles suspended in gelled agarose agarose were lysed with detergent and protease, stained with ethidium bromide and their DNA was observed by fluorescence microscopy. The migration of individual DNA molecules during electrophoresis on a microscope slide was recorded on video tape so that moving pictures could be analyzed. The DNA from lysed bacteria (Escherichia coli andAgrobacterium tumefaciens) appeared as a rosette of at least twenty loops of varying size, whereas that from bacterial spheroplasts (E. coli andPseudomonas aeruginosa) appeared as circular forms or rods with many fine filaments of RNA extending toward the anode. The DNA from chloroplasts of watermelon (Citrullus vulgaris) and pea (Pisum sativum) did not appear as a rosette of loops. Many or most of the chloroplast DNA molecules per lysed chloroplast were immobile in the electric field, as if in circular form hooked on agarose fibers. The amount of DNA-fluorescence per watermelon mitochondrial particle was much less than that found for either chloroplasts or bacteria. The appearance of the mitochondrial DNA during electrophoresis was that of linear molecules, no obviously circular forms were evident and no rosette structures were observed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - kb kilobase pairs - mtDNA mitochondrial DNA - PFGE pulsed-field gel electrophoresis     

The isolation of high molecular weight DNA from wheat,barley and rye for analysis by pulse-field gel electrophoresis

A method is presented for the preparation of large DNA molecules from protoplasts embedded in agarose blocks of three different cereals-hexaploid bread wheat (Triticum aestivum), barley (Hordeum vulgare) and rye (Secale cereale). Pulse-field gel electrophoresis (PFGE) analysis of these DNA preparations using a contour-clamped homogeneous field (CHEF) apparatus indicated that the size of the DNA molecules was greater than 6 Mb. DNA samples prepared by this method were shown to be useful for restriction analysis using both frequent and rare cutting enzymes.     

The Size of DNA Molecules and Chromatin Organization in the Macronucleus of the Ciliate Didinium nasutum (Ciliophora)

Pulsed‐field gel electrophoresis (PFGE) was applied to analyze the molecular karyotype of the ciliate Didinium nasutum. The data obtained indicate that D. nasutum belongs to the ciliate species with subchromosomal macronuclear genome organization. No short “gene‐sized” DNA molecules were detected. Macronuclear DNAs formed a continuous spectrum from 50 kbp to approximately 1,000 kbp in size with a peak plateau between 250 and 400 kbp. The macronuclear DNA molecules were packed into chromatin bodies of 80–265 nm in size. Comparison of the PFGE and electron microscopic data shows that most if not all chromatin bodies contain more than one DNA molecule.     

Pulsed-field gel electrophoresis: a versatile tool for analysis of fungal genomes. A review

The separation of chromosome-size DNA molecules by pulsed-field gel electrophoresis (PFGE) has become a well-established technique in recent years. Although it has very wide-ranging applications, it made a real breakthrough for fungal genome analysis. Because of the small size of fungal chromosomes, their investigation was not possible earlier. Different PFGE approaches allowed the separation of DNA molecules larger than 10 megabase pairs in size, and electrophoretic karyotypes for numerous previously genetically uncharacterized fungal species could be established. This review discusses the applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation.     

Measurement of radiation-induced DNA double-strand breaks by pulsed-field gel electrophoresis

We have examined the use of pulsed-field gel electrophoresis (PFGE) to measure DNA double-strand breaks induced in CHO cells by ionizing radiation. The PFGE assay provides a simple method for the measurement of DNA double-strand breaks for doses as low as 3-4 Gy ionizing radiation, and appears applicable for the measurement of damage produced by any agent producing double-strand breaks. The conditions of transverse alternating field electrophoresis determined both the sensitivity of the assay and the ability to resolve DNA fragments with different sizes. For example, with 0.8% agarose and a 1-min pulse time at 250 V for 18 h of electrophoresis, 0.39% of the DNA per gray migrated into the gel, and only molecules less than 1500 kb could be resolved. With 0.56% agarose and a 60-min pulse time at 40 V for 6 days of electrophoresis, 0.55-0.90% of the DNA per gray migrated into the gel, and molecules between 1500 and 7000 kb could be resolved.     

A method for overcoming DNA degradation during PFGE for Serratia marcescens

The DNA of some bacteria is broken up by Tris-dependent endonuclease activity during the process of sample preparation for pulsed field gel electrophoresis (PFGE). Adding thiourea to the electophoresis buffer for isolates that exhibit DNA degradation has been the method used for many bacterial genera. For a particular group of isolates of Serratia marcescens this method was unsuccessful. A combination of techniques was used to overcome the problem.     

脉冲电泳不同参数对介导GFP基因转化玉米种胚效果的研究

利用脉冲电泳介导绿色荧光蛋白(GFP)基因导入玉米种胚;以GFP基因在种胚中瞬时表达作为外源基因导人种胚细胞的标记,分析了外源．DNA浓度、电泳时间、电压、电流转换时间等脉冲电泳转化参数对种胚发芽率和外源基因导入率的影响。结果表明：脉冲电泳时间对种胚发芽率和外源GFP基因导入率影响最大;通过脉冲电泳可将外源基因导入胚芽细胞,其GFP基因导人种子的频率与各电泳参数均呈二次曲线关系,300μg／ml外源DNA浓度、120min电泳时间、5V电压、2s电流转换时间可作为脉冲电泳介导玉米种胚转化较适宜的参数。     

Randomly distributed DNA double-strand breaks as measured by pulsed field gel electrophoresis: A series of explanatory calculations

The aim of this article is to characterize expressions of relevance to the interpretation of pulsed field gel electrophoresis (PFGE) experiments where randomly distributed double-strand breaks (DSBs) are detected as smears of DNA fragments. Specifically, equations for conversion of percentages of fragments in defined size ranges to DSBs were derived. Several models have been used, one of which is based on theoretically fragmented DNA from the fission yeastSchizosaccharomyces pombe, which has three PFGE separable chromosomes.     

A comparison of molecular typing methods for Moraxella catarrhalis

AIMS: Three molecular typing techniques were examined to determine which method was the most discriminatory in order to perform epidemiological typing of Moraxella catarrhalis. METHODS AND RESULTS: Twenty-five Mor. catarrhalis isolates obtained from nasopharyngeal aspirates collected from Aboriginal and non-Aboriginal children were subjected to random amplified polymorphic DNA (RAPD) analysis, automated ribotyping and pulsed field gel electrophoresis (PFGE). RAPD analysis determined two Mor. catarrhalis types, automated ribotyping with PstI determined four Mor. catarrhalis ribogroups and PFGE analysis with NotI determined 21 pulse field groups within the 25 isolates examined. CONCLUSIONS: Analysis of discrimination index and typeability demonstrated that PFGE is the most discriminatory method for typing Mor. catarrhalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms that PFGE is the most appropriate molecular tool for the epidemiological study of Mor. catarrhalis.     

Evaluation and comparison of random amplification of polymorphic DNA, pulsed-field gel electrophoresis and ADSRRS-fingerprinting for typing Serratia marcescens outbreaks

Amplification of DNA fragments surrounding rare restriction sites (ADSRRS-fingerprinting) is a novel assay based on suppression of polymerase chain reaction (PCR). This phenomenon allows the amplification of only a limited subset of DNA fragments, since only those with two different oligonucleotides ligated at the ends of complementary DNA strands are amplified in the PCR. The DNA fragments can be easily analyzed on polyacrylamide gels, stained with ethidium bromide. We have implemented this method using a set of clinical Serratia marcescens isolates from three outbreaks ongoing in the Public Hospital in Gdańsk (Poland). Clustering of ADSRRS-fingerprinting data matched epidemiological, microbiological, random amplification of polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) data. Based on this study, we found that there is at least a similar power of discrimination between the present 'gold-standard' PFGE and the novel method, ADSRRS-fingerprinting. Although the ADSRRS-fingerprinting method may appear to be more complex than the RAPD technique, we found it fast and reproducible.     

Optimization of pulsed field gel electrophoresis (PFGE) conditions for thermophilic <Emphasis Type="Italic">bacilli</Emphasis>

Although thermophilic members of the genus Bacillus are known to tolerate extreme environmental conditions, they appeared to be readily damaged upon mechanical manipulations. This fact was evidenced in genotyping of some strains of thermophilic Bacillus by pulsed field gel electrophoresis (PFGE). Consequently, a new procedure for the preparation of agarose plugs was developed. The procedure produced interpretable genomic DNA restriction profiles.