Use of haploid mixtures and heteroduplex analysis enhance polymorphisms revealed by denaturing gradient gel electrophoresis

PCR-based codominant genetic markers were developed by using primer sequences designed from cDNA clones of loblolly pine (Pinus taeda L.). Such markers offer certain advantages relative to simple-sequence repeat (SSR), also known as short-tandem repeat (STR) markers, and include the ability to quantify and map DNA polymorphisms in expressed genes. However, detecting these DNA polymorphisms is more problematic because many DNA polymorphisms in genes involve base substitutions rather than insertions or deletions. Denaturing gradient gel electrophoresis (DGGE) is a sensitive and efficient method for detecting sequence differences among PCR fragments. This paper demonstrates the application of DGGE to genetically map expressed genes in loblolly pine. Also, heteroduplex DNA fragments, formed during the amplification of DNA from heterozygotes and from mixes of haploid DNAs from megagametophytes, enhanced and strengthened genetic interpretations and genotypic classifications.     

Culture-independent analysis of probiotic products by denaturing gradient gel electrophoresis

In order to obtain functional and safe probiotic products for human consumption, fast and reliable quality control of these products is crucial. Currently, analysis of most probiotics is still based on culture-dependent methods involving the use of specific isolation media and identification of a limited number of isolates, which makes this approach relatively insensitive, laborious, and time-consuming. In this study, a collection of 10 probiotic products, including four dairy products, one fruit drink, and five freeze-dried products, were subjected to microbial analysis by using a culture-independent approach, and the results were compared with the results of a conventional culture-dependent analysis. The culture-independent approach involved extraction of total bacterial DNA directly from the product, PCR amplification of the V3 region of the 16S ribosomal DNA, and separation of the amplicons on a denaturing gradient gel. Digital capturing and processing of denaturing gradient gel electrophoresis (DGGE) band patterns allowed direct identification of the amplicons at the species level. This whole culture-independent approach can be performed in less than 30 h. Compared with culture-dependent analysis, the DGGE approach was found to have a much higher sensitivity for detection of microbial strains in probiotic products in a fast, reliable, and reproducible manner. Unfortunately, as reported in previous studies in which the culture-dependent approach was used, a rather high percentage of probiotic products suffered from incorrect labeling and yielded low bacterial counts, which may decrease their probiotic potential.     

Online tool for analysis of denaturing gradient gel electrophoresis profiles

We present an online tool (EquiBands, http://www.univie.ac.at/IECB/limno/equibands/EquiBands.html) that quantifies the matching of two bands considered to be the same in different samples, even when samples are applied to different denaturing gradient gel electrophoresis gels. With an environmental example we demonstrate the procedure for the classification of two bands of different samples with the help of EquiBands.     

Dinoflagellate community analysis of a fish kill using denaturing gradient gel electrophoresis

A molecular method using the polymerase chain reaction (PCR) amplification of small subunit gene sequences (18S rDNA) and denaturing gradient gel electrophoresis (DGGE) was used to determine both the population complexity and species identification of organisms in harmful algal blooms. Eighteen laboratory cultures of dinoflagellates, including Akashiwo, Gymnodinium, Heterocapsa, Karenia, Karlodinium, Pfiesteria, and Pfiesteria-like species were analyzed using dinoflagellate-specific oligonucleotide primers and DGGE. The method is sensitive and able to determine the number of species in a sample, as well as the taxonomic identity of each species, and is particularly useful in detecting differences between species of the same genus, as well as differences between morphologically similar species. Using this method, each of eight Pfiesteria-like species was verified as being clonal isolates of Pfiesteria piscicida. The sensitivity of dinoflagellate DGGE is approximately 1000 cells/ml, which is 100-fold less sensitive than real-time PCR. However, the advantage of DGGE lies in its ability to analyze dinoflagellate community structure without needing to know what is there, while real-time PCR provides much higher sensitivity and detection levels, if probes exist for the species of interest, attributes that complement DGGE analysis. In a blinded test, dinoflagellate DGGE was used to analyze two environmental fish kill samples whose species composition had been previously determined by other analyses. DGGE correctly identified the dominant species in these samples as Karlodinium micrum and Heterocapsa rotundata, proving the efficacy of this method on environmental samples. Toxin analysis of a clonal isolate obtained from the fish kill samples confirmed the presence of KmTx2, corroborating the earlier genetic identification of toxic K. micrum in the fish kill water sample.     

Screening for human mitochondrial DNA polymorphisms with denaturing gradient gel electrophoresis

A method involving denaturing gradient gel electrophoresis (DGGE) was developed to detect mitochondrial DNA (mtDNA) polymorphisms in human peripheral T-lymphocytes. DGGE analysis of 100- to 200-bp sequences of low melting temperature domains within the origin/membrane attachment site, NADH dehydrogenase subunit I, cytochrome c oxidase subunit I and two overlapping regions of the tRNA glycine/NADH dehydrogenase subunit III sequences was performed to identify sequence variants at these sites in a human B-cell line TK6 and T-cells from four individuals. A T → C transition at position 16519 in the origin/membrane attachment site in the TK6 cell line and the T-cells from one individual was found. A sequence variant resulting in a G → A transition at position 9966 in the tRNA glycine/NADH dehydrogenase III was identified in another individual. This method should be useful for the rapid screening of polymorphisms in a large number of samples. Received: 19 October 1995 / Revised: 26 March 1996     

Microbial community analysis in crab ponds by denaturing gradient gel electrophoresis

In order to investigate the role of microbial community in aquatic ecology and biogeochemical cycles, the bacterial community in crab ponds was investigated and the effects of aeration and season on the bacterial community were also assessed. Total DNAs from the water samples were amplified with universal primers and the amplicons were then resolved by denaturing gradient gel electrophoresis. Bands from the resulting profiles were excised and sequenced. Cluster analysis of the resulting profiles showed that the microbial community was affected by aeration and season. The microbial community between the surface and bottom of the water was very similar. A total of fifteen bands were obtained in this study. Three of them were 91–99% similar to uncultured bacterium clones. Three were 95–99% similar to uncultured Verrucomicrobia bacteria. Three were 97–100% similar to Actinobacterium sp.. Two were similar to Candidatus Limnoluna rubra with similarity 96 and 99%, respectively. Four were 99% similar to Rhodococcus sp., 100% similar to Sporosarcina sp., 100% similar to Stenotrophomonas sp., and 98% similar to Hydrogenophaga sp., respectively. The concentrations of dissolved oxygen, total nitrogen, total phosphorous, nitrite, and ammonia and pH values were significantly affected by season while only the pH value and the concentrations of dissolved oxygen and total nitrogen were significantly affected by aeration.     

Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.     

Outgroup heteroduplex analysis using temperature gradient gel electrophoresis: high resolution, large scale, screening of DNA variation in the mitochondrial control region

The ability of DNA screening techniques such as Temperature Gradient Gel Electrophoresis (TGGE) and Heteroduplex Analysis to provide resolution approaching that provided by DNA sequencing for a fraction of the time, effort and expense point to them as the logical successor to allozyme electrophoresis for population genetics. Here we present a novel alternative to the standard TGGE/Heteroduplex Analysis protocol - Outgroup Heteroduplex Analysis (OHA). We assess this technique's sensitivity in comparison to previous screening approaches using a known hierarchy of sequence differences. Our data show that Outgroup Heteroduplex Analysis has greatly increased sensitivity for screening DNA variants from that of TGGE used alone and is easily applicable to large numbers of samples. Using this technique we can consistently detect differences of as small as one base change in a 433-base-pair fragment of Control Region mitochondrial DNA from Melomys cerbinipes (an Australian rodent). The approach should easily be extendable to nuclear loci and is not necessarily dependent on the use of a denaturing gradient When combined with a targeted sequencing effort, OHA provides a sensitive and simple means of obtaining allele/haplotype frequencies and their phylogenies for population and phylogeographic studies in molecular ecology.     

用变性梯度凝胶电泳分析PCR克隆的突变率

用变性梯度凝胶电泳技术比较分析了分别由Taq DNA聚合酶和Pfu DNA聚合酶催化扩增的产物克隆入pUCm-T/DH5a系统中产生的重组子,发现包含突变的重组子分别为21．50％和3．50％,前为后的6．15倍。转化为每100nt净扩增长度的突变率分别为7．44％和1．21％。     

Microbial succession during a composting process as evaluated by denaturing gradient gel electrophoresis analysis

Microbial succession during a laboratory-scale composting process of garbage was analysed by denaturing gradient gel electrophoresis (DGGE) combined with measurement of physicochemical parameters such as temperature, pH, organic acids, total dissolved organic carbon and water-soluble humic substance. From the temperature changes, a rapid increase from 25 to 58 degrees C and then a gradual decrease, four phases were recognized in the process as follows; mesophilic (S), thermophilic (T), cooling (C) and maturing (M). The polymerase chain reaction-amplified 16S rDNA fragments with universal (907R) and eubacterial (341F with GC clamp) primers were subjected to DGGE analysis. Consequently, the DGGE band pattern changed during the composting process. The direct sequences from DGGE bands were related to those of known genera in the DNA database. The microbial succession determined by DGGE was summarized as follows: in the S phase some fermenting bacteria, such as lactobacillus, were present with the existing organic acids; in the T phase thermophilic bacillus appeared and, after the C phase, bacterial populations were more complex than in previous phases and the phylogenetic positions of those populations were relatively distant from strains so far in the DNA database. Thus, the DGGE method is useful to reveal microbial succession during a composting process.     

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.     

Comparative analysis of denaturing gradient gel electrophoresis and temporal temperature gradient gel electrophoresis in separating Escherichia coli uidA amplicons differing in single base substitutions

A set of Escherichia coli freshwater isolates was chosen to compare the effectiveness of denaturing gradient gel electrophoresis (DGGE) vs temporal temperature gradient gel electrophoresis (TTGE) for separating homologous amplicons from the respective uidA region differing in one to seven single base substitutions. Both methods revealed congruent results but DGGE showed a five to eight times higher spatial separation of the uidA amplicons as compared with TTGE, although the experiments were performed at comparable denaturing gradients. In contrast to TTGE, DGGE displayed clear and focused bands. The results strongly indicated a significantly higher discrimination efficiency of the spatial chemical denaturing gradient as compared with the temporal temperature denaturing gradient for separating the uidA amplicons. Denaturing gradient gel electrophoresis proved to be highly efficient in the differentiation of E. coli uidA sequence types.     

变性梯度凝胶电泳技术在微生态学研究中应用的进展

变性梯度凝胶电泳(Denaturing gradient gel electrophoresis,DGGE)技术具有无需进行微生物培养、检测率高、分辨率高、重复性好等优点,现已逐渐成为微生态学研究中的强有力工具.本研究介绍了变性梯度凝胶电泳(DGGE)的基本原理和实验步骤中的系统优化,重点介绍和讨论了DGGE在微生态学研究中应用以及进展情况,并对该技术的应用前景进行了展望.     

Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology

Here, the state of the art of the application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology will be presented. Furthermore, the potentials and limitations of these techniques will be discussed, and it will be indicated why their use in ecological studies has become so important.     

Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is believed to be the most powerful pre-screening method for mutation detection currently available, being used mostly on an exon-by-exon basis. Broad-range DGGE for the analysis of multiple fragments or an entire gene is rarely applied. We and others have already shown that one or two DGGE conditions are usually sufficient to analyse an entire gene. Conditions, however, have never been profoundly tested and compared with alternative methods suggested in the literature. Trying to do so in this study, we found significant differences between the various gel systems. The optimal conditions we found for broad-range DGGE include 9% polyacrylamide for the gel, a denaturing gradient with a difference of 30-50% between the lowest and the highest concentration of denaturant, and electrophoresis in 0.5x TAE buffer at a voltage >100 V and <200 V.     

Analysis of fungal communities on historical church window glass by denaturing gradient gel electrophoresis and phylogenetic 18S rDNA sequence analysis.

Besides lichens and bacteria, fungi play a crucial role in the biodeterioration of historical glass. In the present paper, the fungal diversity on the surface of two historical church window glasses was investigated by 18S rDNA-based denaturing gradient gel electrophoresis (DGGE) analysis. 566-bp 18S rDNA-specific clone libraries were constructed with primer set NS1/NS2+10. Positive clones were reamplified with primer sets EF4/518rGC (426-bp fragments) and NS26/518rGC (316-bp fragments), amplicons were screened by DGGE and clustered according to their position in DGGE. Results indicated that fungal 18S rDNA clone libraries should be screened with at least two different primer sets to obtain the maximum number of different clones. For phylogenetic sequence analyses, clone inserts were sequenced and compared with 18S rDNA sequences listed in the EMBL database. Similarity values ranged from 93.7% to 99.81% to known fungi. Analyses revealed complex fungal communities consisting of members and relatives of the genera Aspergillus, Aureobasidium, Coniosporum, Capnobotryella, Engyodontium, Geomyces, Kirschsteiniothelia, Leptosphaeria, Rhodotorula, Stanjemonium, Ustilago, and Verticillium. The genera Geomyces and Aureobasidium were present on both glass surfaces. Some genera had not been detected on historical glass so far.     

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.     

DDGGE, a simplified denaturing gradient gel electrophoresis method for the detection of sequence variations in PCR fragments

The need for rapid analysis of sequence variations in PCR fragments of the same length is increasing in medical diagnostics and environmental studies. Therefore a modified denaturing gradient gel electrophoresis (DGGE) method was developed in which mixed PCR fragments of 1,500 bp could be analysed on a conventional DNA sequencing gel apparatus. In addition, PCR primers without long GC-clamps could be used to amplify the target genes. © Rapid Science Ltd. 1998