变性梯度凝胶电泳装置及其在DNA突变检测中的初步应用

设计了一套适用于变性梯度凝胶电泳(DGGE)的装置。该装置为带缓冲液循环系统、可精确控制温度的夹芯式双垂直电泳槽,其整个凝胶板内的控温精度和均匀度均在±0.5℃范围内。对其在DNA突变检测中的应用作了初步的探讨。通过PCR-DGGE连用成功地分离了野生型p53基因和含点突变的P53基因(第141密码子由TGC突变为TAC)。     

Denaturing gradient gel electrophoresis (DGGE) screening of clones prior to sequencing

Whilst cloning and sequencing techniques are used ubiquitously for the identification of novel DNA sequences, the necessity to determine a consensus sequence means that this can be both labour‐intensive and costly. Here we describe a rapid and cost effective method of using denaturing gradient gel electrophoresis (DGGE) for the analysis of large numbers of clones prior to sequencing. This procedure allows for the selection of specific clones, eliminates the need to sequence multiple copies of the same clone, and reduces the likelihood of sequencing recombinant PCR product.     

Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria

Denaturing gradient gel electrophoresis (DGGE) of PCR amplicons of the ammonia monooxygenase gene (amoA) was developed and employed to investigate the diversity of ammonia-oxidizing bacteria (AOB) in four different habitats. The results were compared to DGGE of PCR-amplified partial 16S rDNA sequences made with primers specific for ammonia-oxidizing bacteria. Potential problems, such as primer degeneracy and multiple gene copies of the amoA gene, were investigated to evaluate and minimize their possible impact on the outcome of a DGGE analysis. amoA and 16S rDNA amplicons were cloned, and a number of clones screened by DGGE to determine the abundance of different motility types in the clone library. The abundance of clones was compared to the relative intensity of bands emerging in the band pattern produced by direct amplification of the genes from the environmental sample. Selected clones were sequenced to evaluate the specificity of the respective primers. The 16S rDNA primer pair, reported to be specific for ammonia-oxidizing bacteria (AOB), generated several sequences that were not related to the known Nitrosospira-Nitrosomonas group and, thus, not likely to be ammonia oxidizers. However, no false positives were found among the sequences retrieved with the modified amoA primers. Some phylogenetic information could be deduced from the position of amoA bands in DGGE gels. The Nitrosomonas-like sequences were found within a denaturant range from 30% to 46%, whereas the Nitrosospira-like sequences migrated to 50% to 60% denaturant. The majority of retrieved sequences from all four habitats with high ammonia loads were Nitrosomonas-like and only few Nitrosospira-like sequences were detected.     

Denaturing gradient gel electrophoresis identifies genomic DNA polymorphism with high frequency in maize

Summary We have used denaturing gradient gel electrophoresis (DGGE) to identify genomic DNA polymorphism in maize (Zea mays L.). DGGE probes detect polymorphism in maize at a frequency comparable to the incidence of restriction fragment length polymorphism (RFLP). Probes identifying polymorphism were mapped to maize chromosome arms by utilizing DGGE and maize lines carrying B-A chromosomal translocations. The methods for library construction, probe screening, and genome analysis, described here for maize, can also be applied to the genomic analysis of other organisms.     

Denaturing gradient gel electrophoresis: a rapid method for differentiating BoLA-DRB3 alleles

The products of the BoLA-DRB3 locus are important molecules in the bovine immune response. Several techniques have been used to study and define this locus but they are generally time consuming and limited in their ability to detect novel alleles. In this study we used denaturing gradient gel electrophoresis (DGGE), and direct sequencing, for BoLA-DRB3 -typing. First, modified locus-specific primers were used in polymerase chain reaction (PCR) to amplify a 240 bp fragment of exon 2 of BoLA-DRB3 from the genomic DNA of 22 cattle and one pair of twin calves. The reverse primer included a GC-rich clamp to improve the physical separation of the BoLA-DRB3 alleles by DGGE. The denaturing gradient needed to produce separation of alleles was determined using perpendicular DGGE, and this gradient was then applied to parallel denaturing gels. The optimal time for producing allele separation was determined using a time-series analysis. The bands representing individual BoLA-DRB3 alleles were excised from the gels, reamplified, and the nucleotide sequence determined using fluorescent-based automated cycle sequencing. The nucleotide sequences of the separated bands were then compared to published BoLA-DRB3 alleles. A gradient of 10–15% acrylamide combined with a 15–50% urea-formamide gradient was successfully used to separate BoLA-DRB3 alleles in all individuals examined. Nucleotide sequencing showed that the 24 animals possessed 13 BoLA-DRB3 alleles, all of which have been previously described. The BoLA-DRB3 genotypes included 20 heterozygotes and two homozygotes. Three BoLA-DRB3 alleles were seen in each of the twin calves, possibly due to leukochimerism. The technique is reliable and rapid, and avoids cloning alleles prior to nucleotide sequencing and therefore offers distinct advantages over previous techniques for BoLA-DRB3 -typing.     

变形梯度胶电泳分析喂养方式对早产新生儿肠道菌群的影响

目的采用变性梯度胶电泳(DGGE)方法研究喂养方式对早产新生儿肠道菌群的影响。方法收集同期6对新生儿1-21d粪便,直接提取细菌总DNA,扩增16SrDNA V6～V8区后DGGE分离,测序并与EMBL核苷序列数据库进行比较。结果喂养前肠道菌群类似,以梭状芽胞杆菌、链球菌和克雷伯菌为主;开奶后母乳喂养儿以双歧杆菌为主,奶粉喂养儿肠道菌群显示其明显的多态性,有双歧杆菌、梭状芽胞杆菌、链球菌、大肠埃希菌、克雷伯菌、韦荣菌、沙雷菌以及不经培养细菌。结论喂养方式对早产新生儿菌群的形成及演替有明显影响,PCR—DGGE在多态性,动力性,菌群的演进变化方面提供了更加准确的数据和补充资料。     

Denaturing gradient gel electrophoresis (DGGE)-based monitoring of intestinal lactobacilli and bifidobacteria of pigs during a feeding trial

The aim of this study was to determine the influence of different feeding strategies on the gut microbiota of organic growing-finishing pigs. A total of 76 pigs were allocated to four different dietary treatments (control, probiotics, maize silage and grass silage). Effects of the applied probiotic preparation on the composition of the intestinal and faecal microbiota were monitored. By using a DGGE (denaturing gradient gel electrophoresis)-based methodology, fingerprints of the intestinal microbiota were obtained. The total microbial DNA was isolated from faecal and colon samples and amplified with PCR using different primer sets to detect bifidobacteria and lactobacilli. PCR products were separated using DGGE and the resulting profiles were compared with the findings of the other dietary treatments. Bands were excised from the gels and sequenced for further identification. Particularly two different DGGE profiles of bifidobacteria were observed, while lactobacilli showed larger variety within the dietary treatments.     

Denaturing gradient gel electrophoresis analysis with different primers of subgingival bacterial communities under mechanical debridement

DGGE of 16S rDNA is one of the most frequently used methods to study microbial communities. In this study, the DGGE profiles of different 16S rDNA regions of the periodontal pathogens Porphyromonas gingivalis, Fusobacterium nucleatum, and Prevotella nigrescens were investigated. The results suggested that V3-V5 and V6-V8 fragments may be suitable for community analysis of subgingival bacteria. Further analysis of subgingival samples with V3-V5 and V6-V8 regions as target fragments suggested that, in chronic periodontitis, re-colonization by periodontal bacteria with a population very similar to the baseline may occur by 6 weeks after mechanical debridement.     

Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota.

The porcine gastrointestinal tract (GIT) microbiota has been studied to increase production efficiency, improve product quality, and help attempt to reduce disease. During the developmental period from birth through weaning, the intestinal microbiota undergoes a rapid ecological succession. There is interest in developing a monitoring technique that allows for analysis of bacterial population levels and shifts within the pig intestine. The objective of this study was to determine if denaturant gradient gel electrophoresis (DGGE) could be effectively applied to measure changes in bacterial populations of the pig GIT, as influenced by age, diet or compartment. Bacterial genetic diversity was determined using DGGE analysis of the V3 region of 16S rDNA PCR products (approximately 200 bp) obtained from primers specific for the domain Bacteria. Protocol development included optimization of: DNA extraction procedures, PCR amplification, removal of PCR artifacts, and optimization of gel preparation and image capture. DGGE analysis revealed diverse bacterial populations between pigs of different ages and among individual gut compartments. Comparison of fecal DNA from different aged pigs revealed several unique PCR product bands indicating the presence of unique bacterial populations. Comparison of different gut compartments demonstrated that bacterial populations were most similar (C, value > 50%) within a single compartment and between adjacent ones. Thus, DGGE can be used to examine bacterial diversity and population shifts in the pig GIT.     

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.     

Use of denaturing gradient gel electrophoresis for analysis of the stool microbiota of hospitalized patients

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal RNA gene amplicons was used to study the stool microbiota of hospitalized patients and to examine the effect of antibiotic therapy. For one patient, 16 anaerobic species identified by random cloning and sequencing of PCR-amplified rRNA genes from stool were represented by bands on the DGGE gel. DGGE analysis and similarity index comparisons demonstrated that the anaerobic microbiota of this individual remained stable in the absence of antibiotic therapy, was minimally affected by ciprofloxacin but markedly reduced by clindamycin therapy, and recovery of some organisms was evident within days after discontinuation of clindamycin. DGGE analysis of additional patients demonstrated similar disruptions of the intestinal microbiota associated with antibiotic therapy. The DGGE banding patterns of nine patients showed considerable variability, but several bands were shared among patients. Thus, our findings are consistent with previous studies that utilized culture techniques, and suggest that DGGE is a useful technique for analysis of the stool microbiota of hospitalized patients.     

Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples

Mangroves are complex ecosystems that regulate nutrient and sediment fluxes to the open sea. The importance of bacteria and fungi in regulating nutrient cycles has led to an interest in their diversity and composition in mangroves. However, very few studies have assessed Archaea in mangroves, and virtually nothing is known about whether mangrove rhizospheres affect archaeal diversity and composition. Here, we studied the diversity and composition of Archaea in mangrove bulk sediment and the rhizospheres of two mangrove trees, Rhizophora mangle and Laguncularia racemosa, using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing of archaeal 16S rRNA genes with a nested-amplification approach. DGGE profiles revealed significant structural differences between bulk sediment and rhizosphere samples, suggesting that roots of both mangrove species influence the sediment archaeal community. Nearly all of the detected sequences obtained with pyrosequencing were identified as Archaea, but most were unclassified at the level of phylum or below. Archaeal richness was, furthermore, the highest in the L. racemosa rhizosphere, intermediate in bulk sediment, and the lowest in the R. mangle rhizosphere. This study shows that rhizosphere microhabitats of R. mangle and L. racemosa, common plants in subtropical mangroves located in Rio de Janeiro, Brazil, hosted distinct archaeal assemblages.     

Denaturing gradient gel electrophoresis (DGGE): a rapid and sensitive technique to screen nucleotide sequence variation in populations

We describe a rapid and sensitive method for the detection of nucleotide sequence variation that can be used for large-scale screening of population markers. Denaturing gradient gel electrophoresis (DGGE) detects sequence variants of amplified fragments by the differences in their melting behavior. DGGE detects most single-base substitutions when carried out on products amplified with a primer to which a GC clamp has been added. Although DGGE has been primarily used for the detection of limited numbers of single-base mutations in disease studies, it offers great potential for use in population analysis of genetic markers with greater levels of sequence variation. The methodology described was developed to identify the number and distribution of MHC class I alpha 1 alleles among chinook salmon (Oncorhynchus tshawytscha) populations. DGGE detects 28 of 31 identified alpha 1 sequences, which differ by between 1 and 16 nucleotides and a two-codon indel. By creating a network of control alleles, 22-23 of the MHC alleles can be resolved rapidly and accurately by a single gel run condition, and 27 alleles can be resolved by two gel run conditions. This techniques has been used in surveys scoring alleles from two MHC markers (class I alpha 1 and alpha 2) in 20,000 individuals of chinook and coho (O. kisutch) salmon. A single person in our laboratory now analyzes 160 salmon from one MHC locus per day with DGGE.     

Denaturing gradient gel electrophoresis analyses of the vertical distribution and diversity of Vibrio spp. populations in the Cariaco Basin

The Cariaco system is the second largest permanently anoxic marine water body in the world. Its water column is characterized by a pronounced vertical layering of microbial communities. The goal of our study was to investigate the vertical distribution and diversity of Vibrio spp. present in the Cariaco Basin waters using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. Representatives of the Vibrio genus were detected by nested and direct PCR in seawater at 10 depths. Sequence analyses of 55 DGGE bands revealed that only 11 different operational taxonomic units (OTU) are identified as Vibrio species. Between one and five OTUs were detected at each depth and the most common OTUs were OTU 1 and OTU 2, which phylogenetically clustered with Vibrio chagasii and Vibrio fortis, respectively. OTUs 3 and 4 were only found in the anoxic zone and were identified as Vibrio orientalis and Vibrio neptunius, respectively. Several Vibrio species detected are potentially pathogenic to human, prawns and corals such as Vibrio parahaemolyticus, Vibrio fischeri and Vibrio shilonii. In the Cariaco Basin, different Vibrio species were found to be specific to specific depths strata, suggesting that this genus is a natural component of the microbial communities in this marine redox environment.     

Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat.

Previous studies investigating microbial diversity in the Octopus Spring cyanobacterial mat community (Yellowstone National Park) have shown a discrepancy between bacterial populations observed by molecular retrieval and cultivation techniques. To investigate how selective enrichment culture techniques affect species composition, we used denaturing gradient gel electrophoresis (DGGE) separation of PCR-amplified 16S rRNA gene fragments to monitor the populations contained within enrichment cultures of aerobic chemoorganotrophic bacteria from the ca. 50 degrees C region of the mat community. By varying the degree of dilution of the inoculum, medium composition, and enrichment conditions and duration and by analyzing the cultures by DGGE, we detected 14 unique 16S rRNA sequence types. These corresponded to alpha-, beta-, gamma-, and delta-proteobacteria, Thermus relatives, and gram-positive bacteria with high G + C ratio and, at the highest inoculum dilutions, Chloroflexus aurantiacus relatives, which were estimated to still be approximately 300 times less abundant than cells of the mat primary producer, Synechococcus spp. Only three of these populations were previously cultivated on solidified medium after similar enrichment. Only two of these population have 16S rRNA sequences which were previously cloned directly from the mat. These results reveal a diversity of bacterial populations in enrichment culture which were not detected by either molecular retrieval or strain purification techniques.     

Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial community composition in deep-sea sediments of the South China Sea

The South China Sea, which is one of the largest marginal seas in the world, is predicted to have suitable accumulation conditions and exporting prospects for natural gas hydrate. The aim of this study was to explore the bacterial community composition of deep-sea sediments from such an ecosystem. DNA was extracted by five different methods and used as templates for PCR amplification of the V3 regions of the 16S rRNA gene. Denaturing gradient gel electrophoresis (DGGE) was used to separate the amplified products and analyse the 16S rRNA gene diversity of sediment samples. The results of DGGE indicated that the bacterial community composition is influenced by DNA extraction methods. Sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belong to Proteobacteria, Bacteroidetes, gram-positive bacteria and Archaea. Integrating different DNA extraction procedures are needed to analyse the actual bacterial diversity from environment when the amplification of 16S rRNA gene and construction of representative clone library were adopted.     

Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community.

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA gene segments was used to profile microbial populations inhabiting different temperature regions in the microbial mat community of Octopus Spring, Yellowstone National Park. DGGE allowed a rapid evaluation of the distributions of amplifiable sequence types. Profiles were essentially identical within regions of the mat defined by one temperature range but varied between sites with different temperature ranges. Individual DGGE bands were sequenced, and the sequences were compared with those previously obtained from the mat by cloning and from cultivated Octopus Spring isolates. Two known cyanobacterial populations and one known green nonsulfur bacterium-like population were detected by DGGE, as were many new cyanobacterial and green nonsulfur and green sulfur bacterium-like populations and a novel bacterial population of uncertain phylogenetic affiliation. The distributions of several cyanobacterial populations compared favorably with results obtained previously by oligonucleotide probe analyses and suggest that adaptation to temperature has occurred among cyanobacteria which are phylogenetically very similar.