Evidence that DNA fragmentation in apoptosis is initiated and propagated by single-strand breaks

Apoptosis is characterised by the degradation of DNA into a specific pattern of high and low molecular weight fragments seen on agarose gels as a distribution of sizes between 50-300 kb and sometimes, but not always, a ladder of smaller oligonucleosomal fragments. Using a 2D pulsed field-conventional agarose gel electrophoresis technique, where the second dimension is run under either normal or denaturing conditions, we show that single-strand breaks are introduced into DNA at the initial stages of fragmentation. Using single-strand specific nuclease probes we further show that the complete fragmentation pattern, including release of small oligonucleosomal fragments can also be generated by a single-strand endonuclease. Three classes of sites where single-strand breaks accumulate were identified. The initial breaks produce a distribution of fragment sizes (50 kb to >1 Mb) similar to those generated by Topoisomerase II inhibitors suggesting that cleavage may commence at sites of attachment of DNA to the nuclear matrix. A second class of rare sites is also cut further reducing the size distribution of the fragments to 50-300 kb. Thirdly, single-strand breaks accumulate at the linker region between nucleosomes eventually causing double-strand scissions which release oligonucleosomes. These observations further define the properties of the endonuclease responsible for DNA fragmentation in apoptosis.     

Two dimensional single-strand conformation polymorphism analysis: a useful tool for the detection of mutations in long DNA fragments.

A new two-dimensional gel system for the analysis of single strand conformational polymorphisms has been developed to identify point mutations, deletions and insertions in long DNA fragments (e.g. 2.7 kb) generated by the polymerase chain reaction. In this procedure, such DNA fragments are first restricted with frequent-cutter enzymes. The resulting small fragments are then separated in the first dimension according to their size by electrophoresis under denaturing conditions; these single stranded DNA fragments are subsequently fractionated in the second dimension by electrophoresis on a non denaturing slab gel based on their fold-back conformation which is completely sequence-dependent. The method was tested on three previously characterized pH 4.5 resistant mutants of HRV14 and was then used to determine changes in three further mutants.     

Alterations in DNA helix stability due to base modifications can be evaluated using denaturing gradient gel electrophoresis

DNA molecules that differ by a single base-pair can be separated by denaturing gradient gel electrophoresis due to the sequence-specific melting properties of DNA. Base modifications such as methylation are also known to affect the melting temperature of DNA. We examined the final position of DNA fragments containing either 5-methyl-cytosine or 6-methyl-adenine in denaturing gradient gels. The presence of a single methylated base within an early melting domain resulted in a well-resolved shift in fragment position relative to the unmethylated sequence. In addition, fragments containing hemimethylated and fully methylated sites could be distinguished, and a proportionally larger shift was observed with an increasing number of methylated bases. Denaturing gradient gel electrophoresis thus provides a sensitive method for analyzing the methylation state of DNA, which is not dependent on the presence of restriction enzyme cleavage sites. We also demonstrate that denaturing gradient gel electrophoresis can be used to obtain a quantitative estimate of the change in helix stability caused by modification of one or two bases in a complex DNA sequence. Such estimates should allow more accurate modeling of melting of natural DNA sequences.     

Genomic distribution of repeated and single copy DNA sequences in Penicillium funiculosum

Study of the reassociation kinetics of Penicillium funiculosum DNA (fragment size > 20 kb) revealed an interspersion of repeated and single-copy DNA sequences. The fragment length of interspersed single-copy DNA was estimated to be more than 9 kb from a curve relating the fraction of DNA binding to hydroxyapatite as a function of DNA fragment length. The length of interspersed short and long repeated nucleotide sequences was determined to be 0.55 kb and > 20 kb, respectively, by agarose gel electrophoresis. The genomic organization of P. funiculosum is thus significantly different from that in other fungi.     

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.     

Separation of fluorescence-labelled terminal restriction fragment DNA on a two-dimensional gel (T-RFs-2D) - an efficient approach for microbial consortium characterization

Fingerprinting techniques provide access to understanding the ecology of uncultured microbial consortia. However, the application of current techniques such as terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) has been hindered due to their limitations in characterizing complex microbial communities. This is due to that different populations possibly share the same terminal restriction fragments (T-RFs) and DNA fragments may co-migrate on DGGE gels. To overcome these limitations, a new approach was developed to separate terminal restriction fragments (T-RFs) of 16S rRNA genes on a two-dimensional gel (T-RFs-2D). T-RFs-2D involves restriction digestion of terminal fluorescence-labelled PCR amplified 16S rRNA gene products and their high-resolution separation via a two-dimensional (2D) gel electrophoresis based on the T-RF fragment size (1(st) D) and its sequence composition on the denaturing gradient gel (2(nd) D). The sequence information of interested T-RFs on 2D gels can be obtained through serial poly(A) tailing reaction, PCR amplification and subsequent DNA sequencing. By employing the T-RFs-2D method, bacteria with MspI digested T-RF size of 436 (±1) bp and 514 (±1) bp were identified to be a Lysobacter sp. and a Dehalococcoides sp. in a polychlorinated biphenyl (PCB) dechlorinating culture. With the high resolution of 2D separation, T-RFs-2D separated 63 DNA fragments in a complex river-sediment microbial community, while traditional DGGE detected only 41 DNA fragments in the same sample. In all, T-RFs-2D has its advantage in obtaining sequence information of interested T-RFs and also in characterization of complex microbial communities.     

Comprehensive detection of single base changes in human genomic DNA using denaturing gradient gel electrophoresis and a GC clamp

We present a simple, efficient extension of denaturing gradient gel electrophoresis that allows the detection of nearly any sequence change in a defined fragment of DNA. The fragment can be obtained either by means of the polymerase chain reaction or by restriction digestion of genomic DNA. With restriction fragments of genomic DNA, sequence information is not required, and covalent modifications in genomic DNA that are lost in a PCR, such as methylation, are detectable. We describe how a GC clamp (an arbitrary, G+C-rich sequence of 30 to 60 bp) can be attached to a selected restriction fragment present in a digest of genomic DNA. The GC clamp alters the melting properties of the fragment; this change greatly increases the fraction of possible mutations that is detectable. In a 272-bp HaeIII fragment from the human beta-globin gene, we were able to detect 13 of 13 mutations tested in human genomic DNA. Four additional mutations in cloned plasmids were analyzed. The data agree with a simple theoretical model for DGGE, which predicts how two fragments, differing at a single (specified) base pair, are resolved in a gradient gel as a function of running time for the gel. The calculation assists in the design of probes and gel conditions that aid in the detection of sequence changes.     

LD-PCR coupled to long-read direct sequencing: an approach for mutation detection in genes with compact genomic structures

A number of techniques have been developed as primary screens to scan for DNA sequence variants, including denaturing gradient gel electrophoresis, denaturing high-performance liquid chromatography, single-strand conformation polymorphism and heteroduplex analysis. Variant alleles detected by these assays are subsequently characterised by DNA sequencing. Sequencing itself is rarely used as a primary screen because of labour intensity, cost, and, upon automation, occasional inaccuracy in identifying heterozygous sites. We have previously developed an approach based on coupling long-distance PCR (LD-PCR) to long-read direct sequencing to allow the detection of mutations in the approximately 1.1 kb exon 3 of MECP2. Our use of dye-labelled primers generated high-quality bi-directional sequence runs > 650 bp and allowed easy discrimination of heterozygous bases. We now describe the application of this approach to the detection of mutations in a considerably larger 6.35 kb LD-PCR fragment spanning 10 exons (exons 32-41) of the structurally complex, but genomically compact, TSC2 gene. In a blind analysis, 15/15 previously characterised mutations were successfully identified using seven overlapping bi-directional sequencing reactions. Our approach of long-read sequencing of long-distance PCR products may allow rapid sequencing of multiple exons of compact genes and may be appropriate as a highly sensitive primary screen for mutations.     

Pulsed field gradient electrophoresis of DNA digested in agarose allows the sizing of the large duplication unit of a surface antigen gene in trypanosomes

Intact chromosome-sized DNA molecules from eukaryotes may be prepared by performing lysis and enzymic deproteinization on cells embedded in agarose [Schwartz and Cantor, Cell 37 (1984), 67-75]. Here we show that DNA prepared by this method may be cut with restriction enzymes, or modified with site-specific methylases and cut by DpnI. As the DNA remains incorporated in the gel matrix, shear degradation of large fragments is avoided. The fragments can then be sized by conventional or pulsed field gradient gel electrophoresis. Phage lambda genomic oligomers are used as size markers, allowing the estimation of fragment sizes up to about 1200 kb. We apply these techniques to show that activation of the telomeric gene encoding variant surface antigen 1.3 in Trypanosoma brucei strain 427, involves the duplication of a DNA segment that starts between 29 and 42 kb upstream of the gene and to assign a chromosomal fragment into which the duplicated 1.3 gene may have transposed.     

红树林土壤细菌群落16S rDNA V3片段PCR产物的DGGE分析

从土壤中抽提微生物总DNA ,直接扩增 16SrDNAV3片段 ,应用变性梯度凝胶电泳 (DGGE)和分子克隆技术分析 16SrDNAV3片段的多态性 ,发现地域因素和红树品种都是影响土壤细菌群落结构的因素。通过对杯萼海桑土壤 16SrDNAV3片段PCR产物两个DGGE条带进行分子克隆、序列测定和Blast分析 ,发现每个DGGE条带包含着许多不同的 16SrDNAV3片段 ,并且其中多数为NCBI未收录的序列。这表明DGGE和克隆技术相结合的方法是研究土壤微生物群落结构的一种可行方法。     

Chemical cleavage of mismatch (CCM) to locate base mismatches in heteroduplex DNA

This protocol describes the use of the chemical cleavage of mismatch (CCM) method to assess whether a region of DNA contains mutations and to localize them. Compared with other mutation-detection techniques (such as single strand-conformation polymorphism (SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and denaturing gradient gel electrophoresis (DGGE)) that detect mutations in short DNA fragments and require highly specific melting temperatures, CCM has a higher diagnostic sensitivity suited to the detection of mutations in tumor genes, and can analyze amplicons < or = 2 kb in length. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies unpaired cytosine and potassium permanganate modifies unpaired thymine. The samples are then incubated with piperidine, which cleaves the DNA backbone at the site of the modified mismatched base. Cleavage products are separated by electrophoresis, revealing the identity and location of the mutation. The CCM method can efficiently detect point mutations as well as insertions and deletions. This protocol can be completed in 10 h.     

动物线粒体DNA分析技术及其应用

结合动物线粒体基因组的最新研究进展,对目前动物线粒体基因序列的分析技术进行了概述,分析了应用较广的限制性片段长度多态性(RFLP)、序列特异性寡核苷酸分析(SSO)、DNA芯片、单链构象多态性(SSCP)、变性梯度凝胶电泳(DGGE)和竞争性寡核苷酸引物延伸(COP)等技术的原理、方法和适用范围,并对它们在线粒体基因的遗传、起源和进化等热点问题中的应用进行了阐述。     

现代生物技术在环境微生物学中的应用:Ⅲ.限制性片段长度多态性分析、变性/温度梯度凝胶电泳和报道基因

这是现代生物技术在环境微生物学中的应用系列综述文章的第三篇 ,讨论限制性片段长度多态性 (RFLP)分析、变性梯度凝胶电泳 (DGGE)和温度梯度凝胶电泳 (TGGE)以及报道基因。     

Genotypic Microbial Community Profiling: A Critical Technical Review

Microbial ecology has undergone a profound change in the last two decades with regard to methods employed for the analysis of natural communities. Emphasis has shifted from culturing to the analysis of signature molecules including molecular DNA-based approaches that rely either on direct cloning and sequencing of DNA fragments (shotgun cloning) or often rely on prior amplification of target sequences by use of the polymerase chain reaction (PCR). The pool of PCR products can again be either cloned and sequenced or can be subjected to an increasing variety of genetic profiling methods, including amplified ribosomal DNA restriction analysis, automated ribosomal intergenic spacer analysis, terminal restriction fragment length polymorphism, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, single strand conformation polymorphism, and denaturing high-performance liquid chromatography. In this document, we present and critically compare these methods commonly used for the study of microbial diversity.     

Detection of a 10 kb DNA replication intermediate in human melanoma cells

DNA replication in human melanoma cells is investigated by lysing the cells in dilute alkali. This lysis condition results in the release from parental DNA of the single-stranded DNA fragments located in active replicating units. The size of the released DNA should theoretically range from that of Okazaki-fragments up to that of the entire replication unit. However, the results showed that the released DNA replication intermediates which are detected range in the size between Okazaki-fragments up to 10 kb DNA fragments. The 10 kb DNA fragments show a discrete appearance in agarose gel electrophoresis. Moreover the kinetic results indicate that the ligation of the 10 kb DNA fragments to form high molecular weight DNA is a late step. A prerequisite for the release of this DNA fragment as a discrete population is that there are gaps in the continuity of the newly synthesized DNA spaced roughly 10 kb away from each other.     

Chromosomes of kinetoplastida

We have compared chromosome-sized DNA molecules (molecular karyotypes) of five genera (nine species) of kinetoplastida after cell lysis and deproteinization of DNA in agarose blocks and size fractionation of the intact DNA molecules by pulsed field gradient (PFG) gel electrophoresis. With the possible exception of Trypanosoma vivax and Crithidia fasciculata, all species have at least 20 chromosomes. There are large differences between species in molecular karyotype and in the chromosomal distribution of the genes for alpha- and beta-tubulin, rRNA and the common mini-exon sequence of kinetoplastid mRNAs. In all cases, the rRNA genes are in DNA that is larger than 500 kb. Whereas T. brucei has approximately 100 mini-chromosomes of 50-150 kb, only few are found in T. equiperdum; T. vivax has no DNA smaller than 2000 kb. As all three species exhibit antigenic variation, small chromosomes with telomeric variant surface glycoprotein genes cannot be vital to the mechanism of antigenic variation. The apparent plasticity of kinetoplastid genome composition makes PFG gel electrophoresis a potentially useful tool for taxonomic studies.     

Periodicity and fragment size of DNA from mouse TLT hepatoma chromatin and chromatin fractions using endogenous and exogenous nucleases

Summary The action of micrococcal nuclease, DNase I and DNase II on mouse TLT hepatoma chromatin revealing the periodicity of its structure as visualized by denaturing and nondenaturing gel electrophoresis, was consistent with the action of these enzymes on other chromatins. Micrococcal nuclease showed a complex subnucleosome fragment pattern based on multiples of 10 base pairs with a prominant couplet at 140/160 base pairs and the absence of the 80 base pair fragment. This couplet of the core and minimal nucleosome fragments was conspicuously present in the mononucleosomes found in the 11S fractions of a glycerol gradient centrifugation. DNase I and II produced a fairly even distribution of a 10 base pair increasing series of fragments to about 180 base pairs, a pattern also repeated in the DNA of nucleosome glycerol-gradient fractions. In limited digestions by these nucleases multinucleosomic DNA fragments are pronounced. These fragment lengths are multiples of an estimated average repeat length of nucleosome DNA of 180 base pairs. The action of the endogenous Mg/Ca-stimulated endonuclease produced only limited cuts in the hepatoma chromatin resulting primarily in multi-nucleosommc DNA fragment lengths and only upon lengthy digestion limited subnucleosomic, 10-base-pair multiple fragments are produced. The putative euchromatin-enriched fractions (50–75S) of the glycerol gradient centrifugation of autodigested chromatin, similarly, contained primarily the multinucleosomic DNA fragment lengths. These results are consistent with our previous electron microscopic demonstration that autodigested chromatin as well as the putative euchromatin-enriched fractions were composed of multinucleosomic chromatin segments containing a full complement of histones.     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

DNA指纹图谱技术在土壤微生物多样性研究中的应用

土壤中的微生物多样性是十分丰富的,传统培养方法对土壤微生物多样性的研究有很大局限性。近年来,各种基于16S rDNA基因的指纹图谱分析技术取得了长足的进步,并广泛应用于土壤微生物多样性的研究。这些技术主要有变性梯度凝胶电泳(DGGE)/温度梯度凝胶电泳(TGGE)、单链构象多态性(SSCP)、随机引物扩增多态性DNA(RAPD)、限制性片段长度多态性(RFLP)和扩增核糖体DNA限制性分析(ARDRA)等。对这些技术近年来在土壤微生物多样性研究领域的应用予以简短综述,并初步探讨未来几年土壤微生物分子生态学发展的方向。     

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.