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: 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.     

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％。     

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.     

Molecular inference of dominant picocyanobacterial populations by denaturing gradient gel electrophoresis of PCR amplified 16S rRNA gene fragments

Quantitative accuracy based on the fluorescent intensity of bands in a denaturing gradient gel electrophoresis (DGGE) profile of polymerase chain reaction (PCR)‐amplified 16S rRNA gene fragments was evaluated for the molecular inference of dominant populations using a cyanobacterial primer pair in a picocyanobacterial community. A serial dilution technique of the template prior to PCR of extracted nucleic acids allowed for elimination of minor strains (less than 10% of the whole cell number) using a cell mixture of three known cultured Synechococcus species with different ratios. When the most abundant strain among the three accounted for more than 80% of the cells, the single band derived from the most abundant one was detected exclusively after the template dilution. In the case of two or three strains evenly distributed in the sample, all strains remained as bands after template dilution. The technique used in the present study was also applied to lake water samples collected from depths of 1 and 5 m on 27 August 1999. The same dominant Synechococcus population was detected in both samples. Thus, the template‐dilution technique prior to PCR is useful to determine dominant picocyanobacterial populations in the DGGE profiling.     

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.     

不同压力条件下油藏内源细菌群落激活过程中变性梯度凝胶电泳分析?

摘要：【目的】了解常压（1 MPa）和高压（10 MPa）条件下内源细菌激活中的细菌群落和结构的变化。【方法】利用胜利油田沾3×24井产出液水样,在常压和高压下进行富集培养后,定时取样,样品用变性梯度凝胶电泳（denature gradient gel electrophoresis,DGGE）技术分析。【结果】通过对内源微生物激活前后DGGE条带的数量和亮度变化分析表明,油藏环境中的细菌在种类和数量上并不丰富,激活剂的加入改变了菌群原有的贫营养环境,从而使一些因营养缺乏生长受抑制细菌得以大量繁殖,菌群结     

Screening of bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis, focussed on Bacillus and related endospore-forming genera

AIMS: To screen for bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis (DGGE). As members of Bacillus and related genera were found to persist in the final product, this study focussed on these taxa. METHODS AND RESULTS: Template DNA was extracted from gelatine samples at five crucial points of a gelatine production process. A primer specific for Bacillus and related genera was designed and used in a selective PCR, followed by a nested DGGE-PCR targeting the V9 region of the 16S rDNA. DGGE analysis of the resulting amplicons, and sequence analysis of selected bands, showed high sequence similarities of these bands with Bacillus fumarioli, B. licheniformis, B. coagulans and Clostridium perfringens. When the selective PCR was omitted, primarily Lactobacillus bands were retrieved. CONCLUSIONS: PCR-DGGE analysis of gelatine extracts can be used for tracing and screening of bacterial contamination during gelatine production. A selective PCR, nested with DGGE-PCR, gave much more accurate information about endospore-forming contaminants than did the direct DGGE procedure alone. Significance AND IMPACT OF THE STUDY: Use of this nested DGGE-PCR protocol may provide important information about possible hazards to the final microbiological quality and/or safety of gelatine, so allowing production parameters and/or remediation procedures may be adjusted on-line.     

Survival of silage lactic acid bacteria in the goat gastrointestinal tract as determined by denaturing gradient gel electrophoresis

Aims: To determine the survival rate of silage lactic acid bacteria (LAB) in the ruminant gastrointestinal tract. Methods and Results: Wilted Italian ryegrass (Lolium multiflorum Lam.) silage (containing 1·9 × 10⁶ CFU LAB g^?1) was fed ad libitum to three goats equipped with rumen cannulae. Silage was given alone or with concentrates at a 1 : 1 ratio on a dry matter basis. Rumen fluid was then obtained 2, 4 and 8 h after the morning feeding. Denaturing gradient gel electrophoresis was performed to compare LAB communities in silage, rumen fluid and faeces. The LAB detected in the wilted silage included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus murinus and Lactobacillus sakei. Bands indicative of Lact. murinus were detected in either the rumen fluid or faeces, whereas the bands indicative of Lact. plantarum, Lact. brevis and Lact. sakei were not. Although the rumen fluid LAB counts and volatile fatty acid concentrations were higher in goats fed silage plus concentrates compared with those fed silage alone, the LAB communities themselves remained unaffected. Sampling times and goat‐to‐goat variations did not affect the LAB communities found in the rumen fluid. Conclusion: LAB communities found in the gut are not remarkably affected by the consumption of silage LAB, even when the silage is accompanied by concentrates that facilitate gut fermentation. Significance and Impact of the Study: Although silage can improve probiotic function, it may be difficult for silage LAB to survive the digestive process in the ruminant gastrointestinal tract.     

Changes in the diversity of pig ileal lactobacilli around weaning determined by means of 16S rRNA gene amplification and denaturing gradient gel electrophoresis

Our study aimed to provide a comprehensive characterization of changes in porcine intestinal Lactobacillus populations around the time of weaning based on 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE). DNA was extracted from the ileal contents of piglets at weaning (28 days of age) and after 1, 2, 5 and 11 days. PCR amplicons (V2-V3 fragments of 16S rRNA genes) were separated using DGGE. Predominant bands were excised and sequenced after reamplification. A band corresponding to Lactobacillus salivarius was present 1 and 2 days post-weaning (pw), while Lactobacillus crispatus was detected only 1 and 11 days pw. Lactobacillus sobrius gave the most dominant band in all animals. The number of bands decreased from 13+/-3 at weaning to 9+/-1 at 5 days pw, but the species richness had recovered by 11 days pw. The similarity of profiles between sampling days was high for 1 and 2 days pw (>91%), but was low for 5 and 11 days pw (<59%). The diversity of the profiles was lower 5 days pw, based on the Shannon diversity index (0.83+/-0.076 vs. 1.02+/-0.127 at weaning, P=0.042), but had recovered to preweaning values by 11 days pw. The application of group-specific DGGE showed that the Lactobacillus community within the porcine ileum undergoes dramatic, partly reversible changes as a consequence of weaning.     

Variations in bacterial communities in laboratory-scale and big bale silos assessed by fermentation products, colony counts and denaturing gradient gel electrophoresis profiles

Aims: To assess the variation in bacterial communities in laboratory‐scale and big bale silos. Methods and Results: Wilted Italian ryegrass (628 g dry matter kg^?1) was ensiled in vacuum‐packed plastic pouches and big bales. Silos were opened after 3 months, and the fermentation products, colony counts and denaturing gradient gel electrophoresis (DGGE) profiles were determined. Eight samples were collected separately from a big bale, while one representative sample was taken from a plastic pouch. Significant variation was found between big bales in dry matter, ethanol, lactic acid, acetic acid and ammonia‐N contents. No differences were shown between plastic pouches and big bales, except that more ethanol was produced in the former air‐tight silos. Plastic pouches could resemble a specific silo and outer sampling sites of big bales based on fermentation products and DGGE profiles respectively. Conclusions: Considerable variation in fermentation products may exist between big bale silos. Plastic pouches can serve as a model of big bale silos, although they do not provide information on the heterogeneity within and between bales. Significance and Impact of the Study: Assessment of bacterial communities associated with ensiling can differ according to the criteria of fermentation products, colony counts and DGGE profiles.     

利用变性梯度凝胶电泳分析微生物的多样性

综述了不依赖于培养的变性梯度凝胶电泳技术 (DGGE)分析微生物多样性的原理 ,并列举它的应用实例。DGGE和传统方法相比有很多优点 ,若将DGGE和其他方法结合起来 ,效果更好 ,应用更广泛。     

Temperature gradient gel electrophoresis: detection of a single base substitution in the cattle β-lactoglobulin gene

An A in equilibrium with G transition in exon III is known to differentiate alleles A and B of the cattle beta-lactoglobulin (BLG) gene. A BLG exon III fragment containing the transition site was amplified by the polymerase chain reaction. Temperature gradient gel electrophoresis (TGGE) was then used to detect this transition and hence to genotype cattle: the AT base-pair in allele A was readily distinguished from the GC base-pair of allele B. TGGE can be used to detect any single base-pair substitution, and thus is a powerful method of detecting genetic variability.     

Evaluation of the impact of DNA extraction methods on BAC bacterial community composition measured by denaturing gradient gel electrophoresis

Aims: The impact of DNA extraction methods on biological activated carbon (BAC) DNA yield and bacterial community was evaluated. Methods and Results: Three different DNA extraction methods were compared: method a, method b and method c. Method c with ultrasonic pretreatment improved cell lysis efficiency (from 34% to 87%) and DNA yield [from 10·58 μg g^?1 (dry wt) of carbon to 21·42 μg g^?1 (dry wt) of carbon]. denaturing gradient gel electrophoresis profiles obtained by method c recovered the five seeded bacteria (Bacillus subtilis Strain WSO 6, Pseudomonas putida Strain WSO 7, Acinetobacter lwoffii WSO 10, Pseudomonas pertucinogena WSO 11 and Brevibacterium mcbrellneri WSO 13). Conclusions: The results showed method c with ultrasonic pretreatment was the most successful for the analysis of BAC bacterial community because it was effective in the detachment of bacteria and cell lysis, thereby resulting in good yields. Significance and Impact of Study: These results must be taken into consideration when extracting DNA for analysing BAC bacterial community.     

Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments

Specific amplification of 16S rRNA gene fragments in combination with denaturing gradient gel electrophoresis (DGGE) was used to generate fingerprints of Chromatiaceae, green sulfur bacteria, Desulfovibrionaceae, and β-Proteobacteria. Sequencing of the gene fragments confirmed that each primer pair was highly specific for the respective phylogenetic group. Applying the new primer sets, the bacterial diversity in the chemoclines of a eutrophic freshwater lake, a saline meromictic lake, and a laminated marine sediment was investigated. Compared to a conventional bacterial primer pair, a higher number of discrete DGGE bands was generated using our specific primer pairs. With one exception, all 15 bands tested yielded reliable 16S rRNA gene sequences. The highest diversity was found within the chemocline microbial community of the eutrophic freshwater lake. Sequence comparison revealed that the six sequences of Chromatiaceae and green sulfur bacteria detected in this habitat all represent distinct and previously unknown phylotypes. The lowest diversity of phylotypes was detected in the chemocline of the meromictic saline lake, which yielded only one sequence each of the Chromatiaceae, β-2-Proteobacteria, and Desulfovibrionaceae, and no sequences of green sulfur bacteria. The newly developed primer sets are useful for the detection of previously unknown phylotypes, for the comparison of the microbial diversity between different natural habitats, and especially for the rapid monitoring of enrichments of unknown bacterial species. Received: 22 January 1999 / Accepted: 28 April 1999     

Comparison of microbial communities in four different composting processes as evaluated by denaturing gradient gel electrophoresis analysis

AIMS: We aimed to systematically understand the composting processes by a comparison of microbial communities during four full-scale composting processes. METHODS AND RESULTS: Microbial communities during the four different full-scale composting processes were analysed by denaturing gradient gel electrophoresis combined with measurement of physicochemical parameters. Two composting processes utilized sewage sludge and two utilized food-waste. Comparison of the four processes indicated that the concentration of dissolved organic carbon was higher in the food-waste-composting than in the sewage-sludge-composting processes, and microbial communities varied with composting substrate. The tendency for different microbes to appear in the composting process with different concentrations of dissolved organic carbon agreed with a previous study that showed that microbial succession occurred with a decrease in dissolved organic carbon in a laboratory-scale food-waste-composting process. CONCLUSIONS: Our results suggested that the main factor affecting microbial communities in the composting process is the concentration of dissolved organic materials. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to studying microbial communities involved in composting, this research is also the first to study composting mechanisms using molecular methods. The results of our studies may be helpful in the design and management of composting processes.     

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri , Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii . Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.     

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. andEthanologenbacterium sp.), β-proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase ofEthanologenbacterium sp.,Clostridium sp. and uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types ofClostridium sp.Acidovorax sp.,Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the cometabolism of microbial community played a great role of biohydrogen production in the reactors.