Pellicle associated adherence film above incubation broth surface - an inexpensive adjunct to recognizing Candida krusei in the laboratory

Background

Targeting Induced Local Lesions in Genomes (TILLING) is a high throughput reverse genetics tool which detects mismatches (single point mutations or small indels) in large number of individuals of mutagenized populations. Currently, TILLING is intensively used for genomics assisted molecular breeding of several crop plants for desired traits. Most commonly used platform for mutation detection is Li-COR DNA Analyzer, where PCR amplified products treated with single strand mismatch specific nuclease are resolved on denaturing gels. The molecular size of any cut product can be easily estimated by comparing with IR dye labeled markers of known sizes. Similar fluorescent dye labeled size markers are also used for several genotyping experiments. Currently, commercially available size standards are expensive and are restricted up to only 700 bp which renders estimation of products of sizes greater than 700 bases inaccurate.

Findings

A simple protocol was developed for labeling 5' end of multiple DNA size markers with fluorescent dyes. This method involves cloning a pool of different size markers of DNA in a plasmid vector. PCR amplification of plasmid using IR dye labeled universal primers generates 5' fluorescent labeled products of various sizes. The size of products constituting the ladder can be customized as per the need. The generated size markers can be used without any further purification and were found to be stable up to one year at -20°C.

Conclusions

A simple method was developed for generating fluorescent dye labeled size standards. This method can be customized to generate different size standards as per experimental needs. The protocol described can also be adapted for developing labeled size standards for detection on platforms other than Li-COR i.e. other than infra red range of the spectrum.     

Multiple-locus variable-number tandem repeat analysis of Legionella pneumophila using multi-colored capillary electrophoresis

Several methods for typing of Legionella pneumophila exist, one of which is an 8-locus variable-number of tandem repeats analysis (MLVA). This method is based on separating and sizing amplified VNTR PCR products by agarose gel electrophoresis. In the present work, the existing L. pneumophila MLVA-8 assay is adapted to capillary electrophoresis. The assay was multiplexed by using multiple fluorescent labeling dyes and tested on a panel of L. pneumophila strains with known genotypes. The results from the capillary electrophoresis-based assay are shown to be equivalent to, and in a few cases more sensitive than, the gel-based genotyping assay. The assay presented here allows for a swift, automated and precise typing of L. pneumophila from patient or environmental samples and represents an improvement over the current gel-based method.     

重要肠道病原菌多重PCR-基因芯片检测方法研究

目的:建立并初步评价一种针对重要肠道病原菌的多重PCR 基因芯片检测方法。方法：对筛选出的特异引物进行多重PCR优化,将引物分别按种属内混合和种属间混合的方案排查引物间的竞争性抑制现象,再将不同菌属的模板混合,用相对应的混合引物扩增,探寻高效特异的引物组合。分别掺入和不掺入荧光素,验证其对混合PCR反应的影响,并与芯片杂交,探寻多重PCR扩增效率对芯片杂交的影响。分析不同数量引物组合产生的杂交结果,筛选出无交叉反应的最优引物组合。结果:种属内引物混合均得到特异性扩增结果。种属间混合霍乱弧菌和空肠弯曲菌得到部分预期条带,随着混合引物数量的增加,交叉抑制现象也增多。杂交信号强度随多重PCR扩增效率的增加而增强。反应中掺入荧光素的扩增条带产量低于无荧光素的产物。可将35对混合引物拆成3个体系分别标记样品,以避免假阴性结果。结论:PCR反应中掺入荧光素降低扩增效率和杂交效率,但并不影响对杂交结果的判读和数据分析。基因芯片杂交信号强度取决于多重PCR的扩增效率。肠道病原菌多重PCR 基因芯片检测方法具有较高的特异性,混合PCR可以分别按照种属内和种属间的引物组合方案用于多病原的筛检。该基因芯片检测可以采用3个引物体系完成样品标记。     

The kinetics of fluorescent DNA labeling using PCR with different Taq polymerases depends on the chemical structures of modified nucleotides

The kinetics of DNA labeling during PCR using six fluorescent derivatives of 2′-deoxyuridine 5′-triphosphate has been studied. These compounds differ in their chemical structure, total electric charge and the length of the linker between a dye and the C5 position of a pyrimidine base. The efficiency of the incorporation of the fluorescent derivatives into a growing DNA chain by four commercially available Taq DNA polymerases with 5′→3′ exonuclease and hot start activity has been determined using real-time PCR with a TaqMan probe and the subsequent electrophoretic analysis of the reaction products. Modified deoxyuridines with a total positive or negative charge of the chromophore were practically not incorporated by Taq polymerases during PCR. The modified deoxyuridines with a neutral charge of the chromophore were effectively incorporated into DNA. The extended length of the linker between the pyrimidine base and the chromophore led to a lower PCR inhibition and a more effective inclusion of modified nucleotides in the growing DNA chain. This fact can be explained by the reduced steric effects that were caused by the dye. As a result, the most promising combinations of fluorescently labeled nucleotide and Taq polymerase have been chosen for further use in fluorescent DNA labeling.     

Comparison of different labeling methods for the production of labeled target DNA for microarray hybridization

Different labeling methods were studied to compare various approaches to the preparation of labeled target DNA for microarray experiments. The methods under investigation included a post-PCR labeling method using the Klenow fragment and a DecaLabel DNA labeling kit, the use of a Cy3-labeled forward primer in the PCR, generating either double-stranded or single-stranded PCR products, and the incorporation of Cy3-labeled dCTPs in the PCR. A microarray that had already been designed and used for the detection of microorganisms in compost was used in the study. PCR products from the organisms Burkholderia cepacia and Staphylococcus aureus were used in the comparison study, and the signals from the probes for these organisms analyzed. The highest signals were obtained when using the post-PCR labeling method, although with this method, more non-specific hybridizations were found. Single-stranded PCR products that had been labeled by the incorporation of a Cy3-labeled forward primer in the PCR were found to give the next highest signals upon hybridization for a majority of the tested probes, with less non-specific hybridizations. Hybridization with double-stranded PCR product labeled with a Cy3-labeled forward primer, or labeled by the incorporation of Cy3-labeled dCTPs resulted in acceptable signal to noise ratios for all probes except the UNIV 1389a and Burkholderia genus probes, both located toward the 3' end of the 16S rRNA gene. The comparison of the different DNA labeling methods revealed that labeling via the Cy3-forward primer approach is the most appropriate of the studied methods for the preparation of labeled target DNA for our purposes.     

Modified inter-simple sequence repeat PCR protocol for use in conjunction with the LI-COR Gene ImagIR2 DNA analyzer

Inter-simple sequence repeat (inter-SSR) PCR was assessed for use in variety testing of chrysanthemum. This method was modified to allow detailed analysis of DNA profiles on a LI-COR Gene ImagIR2 DNA analyzer system. Protocols for unlabeled PCR were unsuccessful in producing labeled products when using infrared (IR) dye-labeled primers. Various modifications to the known protocols were investigated: (i) different ratios of labeled to unlabeled primer; (ii) various annealing temperatures; (iii) the use of an IR genotyping kit; (iv) end labeling; and (v) direct incorporation and cycle labeling. Successful amplification using labeled primers only occurred when two consecutive reactions were performed. The first PCR was performed using standard protocols for unlabeled reactions. The second PCR used a dilution of these reaction products as a template and 50% IR-labeled and unlabeled primer. The complete procedure leading to a high-resolution analysis of inter-SSR PCR products on a LI-COR system is reported for the first time. This system allows high-throughput fingerprinting with the potential for applications on a commercial scale.     

Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling

The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.     

Mass spectrometry provides accurate characterization of two genetic marker types in Bacillus anthracis

Epidemiological and forensic analyses of bioterrorism events involving Bacillus anthracis could be improved if both variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs) could be combined on a single analysis platform. Here we present the use of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) to characterize 24 alleles from 6 VNTR loci and 11 alleles from 7 SNP loci in B. anthracis. The results obtained with ESI-FTICR-MS were consistent with independent results obtained from traditional approaches using electrophoretic detection of fluorescent products. However, ESI-FTICR-MS improves on the traditional approaches because it does not require fluorescent labeling of PCR products, minimizes post-PCR processing, obviates electrophoresis, and provides unambiguous base composition of both SNP and VNTR PCR products. In addition, ESI-FTICR-MS allows both marker types to be examined simultaneously and at a rate of approximately 1 sample per min. This technology represents a significant advance in our ability to rapidly characterize B. anthracis isolates using VNTR and SNP loci.     

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.     

Persistence and growth of fecal Bacteroidales assessed by bromodeoxyuridine immunocapture

Extraintestinal growth of fecal bacteria can impair accurate assessment of watershed health. Anaerobic fecal bacteria belonging to the order Bacteroidales are attractive candidates for fecal source tracking because they have host-specific distributions and do not grow well in the presence of high oxygen concentrations. Growth of general and human-specific fecal Bacteroidales marker organisms in environmental samples (sewage) and persistence of the corresponding genetic markers were investigated using bromodeoxyuridine (BrdU) DNA labeling and immunocapture, followed by PCR detection. Background amplification of unlabeled controls occasionally occurred when a high number of PCR cycles was used. By using fluorescent detection of PCR products obtained after 15 cycles, which was determined to be quantitative, we enriched for BrdU-labeled DNA and did not detect unlabeled DNA. By using pure cultures of Bacteroides vulgatus, the ability of Bacteroidales bacteria to take up and incorporate BrdU into nascent DNA was confirmed. Fecal Bacteroidales organisms took up and incorporated BrdU into DNA during growth. In sewage incubated aerobically at the in situ temperature, Bacteroidales genetic marker sequences persisted for at least 24 h and Bacteroidales fecal bacteria grew for up to 24 h as well. Detection by PCR using a low, quantitative cycle number decreased the sensitivity of the assay such that we were unable to detect fecal Bacteroidales human-specific marker sequences in unlabeled or BrdU-labeled fractions, even when fluorescent detection was used. Using 30 PCR cycles with unlabeled fractions, human-specific Bacteroidales sequences were detected, and they persisted for up to 24 h in sewage. These data support the utility of BrdU labeling and immunocapture followed by length heterogeneity PCR or fluorescent detection using low numbers of PCR cycles. However, this method may not be sensitive enough to identify cells that are present at low densities in aquatic environments.     

Incorporation of reporter molecule-labeled nucleotides by DNA polymerases. II. High-density labeling of natural DNA

The modification of nucleic acids using nucleotides linked to detectable reporter or functional groups is an important experimental tool in modern molecular biology. This enhances DNA or RNA detection as well as expanding the catalytic repertoire of nucleic acids. Here we present the evaluation of a broad range of modified deoxyribonucleoside 5′-triphosphates (dNTPs) covering all four naturally occurring nucleobases for potential use in DNA modification. A total of 30 modified dNTPs with either fluorescent or non-fluorescent reporter group attachments were systematically evaluated individually and in combinations for high-density incorporation using different model and natural DNA templates. Furthermore, we show a side-by-side comparison of the incorporation efficiencies of a family A (Taq) and B (Vent_R exo^–) type DNA polymerase using the differently modified dNTP substrates. Our results show superior performance by a family B-type DNA polymerase, Vent_R exo^–, which is able to fully synthesize a 300 bp DNA product when all natural dNTPs are completely replaced by their biotin-labeled dNTP analogs. Moreover, we present systematic testing of various combinations of fluorescent dye-modified dNTPs enabling the simultaneous labeling of DNA with up to four differently modified dNTPs.     

The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes

The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The near-infrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip.

     

Effect of highly fragmented DNA on PCR.

We characterized the behavior of polymerase chain reactions (PCR) using degraded DNA as a template. We first demonstrated that fragments larger than the initial template fragments can be amplified if overlapping fragments are allowed to anneal and extend prior to routine PCR. Amplification products increase when degraded genomic DNA is pretreated by polymerization in the absence of specific primers. Secondly, we measured nucleotide uptake as a function of template DNA degradation. dNTP incorporation initially increases with increasing DNA fragmentation and then declines when the DNA becomes highly degraded. We demonstrated that dNTP uptake continues for >10 polymerization cycles and is affected by the quality and quantity of template DNA and by the amount of substrate dNTP. These results suggest that although reconstruction of degraded DNA may allow amplification of large fragments, reconstructive polymerization and amplification polymerization may compete. This was confirmed in PCR where the addition of degraded DNA reduced the resultant product. Because terminal deoxynucleotidyl transferase activity of Taq polymerase may inhibit 3' annealing and restrict the length of template reconstruction, we suggest modified PCR techniques which separate reconstructive and amplification polymerization reactions.     

Fluorophore-quencher pair for monitoring protein motion

A fluorophore/quencher pair capable of detecting conformational changes of DNA-protein complexes is described. The system employs a fluorescent nucleoside analog 1,3-diaza-2-oxophenothiazine (tC) within duplex DNA and a non-fluorescent quencher (TEMPO) attached to an engineered cysteine residue of the protein. The straightforward labeling methodology allows for the placement of the fluorophore and quencher moieties at specific positions suited to studying the conformational change of interest. To illustrate the utility of the tC-TEMPO pair, we have monitored nucleotide-induced conformational changes of the Klenow fragment (KF) polymerase bound to duplex DNA. In this system, tC was incorporated in the primer strand of the duplex, adjacent to the 3′ end, while TEMPO was positioned at the end of the O-helix within the fingers domain of KF. Using steady-state fluorescence spectroscopy, we measured the quenching efficiency in a binary complex of tC-modified DNA and TEMPO-labeled KF and in ternary complexes containing cognate or non-cognate dNTP substrates. The quenching efficiency is significantly enhanced in the presence of a cognate dNTP, indicating that the O-helix has moved closer towards the DNA. In contrast, no significant tC quenching is observed in the presence of a non-cognate dNTP, indicating that the O-helix remains in a position that is beyond the distance reporting range of the tC-TEMPO pair. These results demonstrate that a cognate dNTP substrate induces a large conformational change of the O-helix, which can be sensitively detected using the tC-TEMPO pair. This fluorophore/quencher pair may be useful to study conformational changes associated with other DNA-enzyme complexes.     

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.     

荧光标记复合PCR扩增微卫星位点的构建与优化

本研究采用毛细管电泳技术,构建并优化了荧光标记复合PCR同时扩增多个微卫星位点。主要过程为：首先根据设计所扩增微卫星位点的期望长度,将9个微卫星位点分成两组,5个位点用FAM（蓝色）标记,4个位点用HEX（绿色）标记;两种荧光类型分组优化,用琼脂糖胶电泳检测。其次,荧光标记的复合PCR扩增8个中华绒螯蟹样品的9个微卫星位点,采用ABI3730xl毛细管电泳检测,以ROX500（红色）为长度标准物,结果经Genemapper3.5软件 分析,检测结果表明毛细管电泳检测荧光标记复合PCR产物不仅精确读取微卫星位点的长度（分辨率高达1bp）,还能区分微卫星位点复制时滑链所引起的“回声斑”;调整各微卫星位点引物比列使所有位点扩增强弱均匀。最后,逐一检测复合PCR基本参数（dNTP浓度、 PCR程序和模版DNA用量）对复合PCR产物的影响,优化PCR。结果表明通过毛细管电泳检测荧光标记复合PCR产物来读取微卫星位点的基因型具有精确性、高效性和稳定性。     

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Fusion PCR and gene targeting in Aspergillus nidulans

We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.     

Estimation of [32P]deoxyribonucleoside triphosphates in cell extracts using periodate treatment.

Radioisotopic labeling of nucleotides in cells before extraction, coupled with two-dimensional chromatography on PEI-cellulose¹ thin layers (1,2), has been successfully used in the estimation of rNTP, ADP, and GDP (3,4). This procedure has however been less successful for the dNTP, because the large amounts of rNTP tend to overlap the dNTP so that no system for the two-dimensional development of the chromatograms has been found to give consistently satisfactory separation of all four dNTP from contaminating rNTP. Yegian (5) overcame this difficulty by an initial oxidation of the ribonucleoside compounds in the extract with periodate followed by a fairly complex one-dimensional chromatographic procedure to isolate the dNTP. We have used the initial oxidation with periodate followed by the two-dimensional procedure referred to above (1) and applied the method successfully to extracts of bacterial and mammalian cells in liquid culture.