Adaptation of conformation-sensitive gel electrophoresis to an ALFexpress DNA sequencer to screen BRCA1 mutations

Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mutations with accuracy. This is important in genes such as BRCA1 and BRCA2, in which alterations span the entire gene. We have adapted the CSGE assay to a fluorescent platform--a DNA sequencer one-color technology--that reduces the time involved and enhances resolving power for the complete scanning of the BRCA genes. Electrophoresis has high sensitivity and is performed in less than three hours, and the gel does not require staining with ethidium bromide. Eighteen single-base and six frameshift mutations in the BRCA1 gene were analyzed. We compared the manual and fluorescent CSGE methods, and all mutations were detected with accuracy.     

Rapid detection of DNA sequence variants by conformation-sensitive capillary electrophoresis

The identification of novel sequence variants, which may be either disease-causing mutations or silent polymorphisms, in large numbers of samples is becoming the rate-limiting step in associating diseases with specific genes. This is particularly true in light of the imminent arrival of the complete reference sequence of the human genome. A number of techniques have been developed to analyze DNA samples for sequence variants rapidly. We describe a new technique, capillary-based conformation-sensitive gel electrophoresis (capillary CSGE) that transfers mutation detection from acrylamide gel to capillary electrophoresis. Capillary CSGE was able to detect 7/7 short insertion/deletions and 16/22 base substitutions in a series of random single-nucleotide polymorphisms and known variants in the lipoprotein lipase and BRCA2 genes. This technique has the potential to screen many megabases of DNA in a single day.     

Evaluating mixed samples as a source of error in non-invasive genetic studies using microsatellites

The use of noninvasive genetic sampling (NGS) for surveying wild populations is increasing rapidly. Currently, only a limited number of studies have evaluated potential biases associated with NGS. This paper evaluates the potential errors associated with analysing mixed samples drawn from multiple animals. Most NGS studies assume that mixed samples will be identified and removed during the genotyping process. We evaluated this assumption by creating 128 mixed samples of extracted DNA from brown bear (Ursus arctos) hair samples. These mixed samples were genotyped and screened for errors at six microsatellite loci according to protocols consistent with those used in other NGS studies. Five mixed samples produced acceptable genotypes after the first screening. However, all mixed samples produced multiple alleles at one or more loci, amplified as only one of the source samples, or yielded inconsistent electropherograms by the final stage of the error-checking process. These processes could potentially reduce the number of individuals observed in NGS studies, but errors should be conservative within demographic estimates. Researchers should be aware of the potential for mixed samples and carefully design gel analysis criteria and error checking protocols to detect mixed samples.     

Mutational analysis of the rhodopsin gene in Chinese ADRP families by conformation sensitive gel electrophoresis

Retinitis pigmentosa is a very heterogeneous group of retinal degenerations, with multiple genes identified in each mode of inheritance. For autosomal dominant retinitis pigmentosa (ADRP), the most common gene is the rhodopsin (RHO) gene, mutations in which contribute to about 25% of ADRP in Caucasian population. To investigate the frequency and pattern of RHO point mutations in Chinese patients with ADRP, we have screened the five coding exons and splice sites of the RHO gene in 50 unrelated probands from Chinese ADRP families and 100 normal controls to identify disease-associated mutations, using conformation sensitive gel electrophoresis (CSGE) and direct DNA sequencing. Two RHO mutations, Pro347Leu and Pro327 (1-bp del), were identified each in one family, thus the frequency of RHO mutations among ADRP families in this study is less than 14% (2/50=4%, 95% confidence interval: 1-14%), lower than that in Europe and North America, which may reflect an ethnic difference between Chinese and Caucasian populations. Loss of all phosphorylation sites at the C-terminus and a highly conserved sequence QVS(A)PA may occur because of Pro327(1-bp del). CSGE was found to be a sensitive, simple and practical method for the screening of a large number of samples under highly reproducible conditions, and could be utilized in routine molecular diagnostic laboratories.     

Standardization of automated cell-based protocols for toxicity testing of biomaterials

Advances in high-throughput screening (HTS) instrumentation have led to enormous reduction of costs (e.g., of pipetting stations) and to the development of smaller instruments for automation of day-to-day routines in small research laboratories. In the biomaterials community, there has been an increasing interest for standardized screening protocols to identify cell type-specific cytocompatible biomaterials suitable for tissue engineering (TE) applications. In this study, the authors established a multiplexed assay protocol for toxicity screening of biomaterials using a low- to medium-throughput robotic liquid handling station (LHS). The protocol contains analysis of viability, cytotoxicity, and apoptosis combined in one assay. This study includes performance results of a side-by-side comparison of the EpMotion 5070 LHS and conventional pipetting/dispensing systems. Critical parameters were optimized each for a given platform. Higher accuracy and reproducibility were achieved for LHS compared to manually treated samples. The practicability and accuracy of the method in a typical small laboratory setting were tested by running daily routine tasks by trained and untrained laboratory staff. In addition, advantages and disadvantages as well as the step-by-step application protocol are reported. The approach described provides a potential utility in screening biomaterials toxicity, allowing researchers to meet the needs of low- and medium-throughput laboratories.     

Methods for reverse genetic screening in zebrafish by resequencing and TILLING

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.     

Comparative 2-DE protein analysis in a 3-D geometry gel

Two-dimensional gel electrophoresis (2-DE) separation has not been considered suitable for large-scale comparative protein expression studies due to its limited throughput. We present a high-throughput analysis method based on three-dimensional (3-D) geometry gel electrophoresis. Following conventional isoelectric focusing (IEF), up to 36 immobilized pH gradient (IPG) strips are arrayed on the top surface of a 3-D gel body, and the samples transferred electrokinetically to the gel. A specific thermal management ensures that sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) occurs under identical electrophoretic and thermal conditions, avoiding gel-to-gel variations and thereby providing immediate comparability of the separation patterns. Proteins are Cy3-labeled for online detection of laser-induced fluorescence (LIF). Images are acquired by a digital camera and recorded as a 3-D image stack during electrophoresis. Image processing software decomposes the 3-D image stack into vertical sections representing conventional 2-DE slab gels, making results immediately accessible without further gel processing. The large number of simultaneously analyzed samples (n = 36) allows treating the sample index as a quasi-continuous experimental parameter (e.g., concentration, time, dose). The method offers a wide range of applications in molecular discovery, clinical diagnosis, pharmacology, and toxicology, like protein monitoring during disease development and screening of drug candidates for their effect on protein expression.     

LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis

DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.     

Identifying patterns of DNA for tumor diagnosis using capillary electrophoresis-amplified fragment length polymorphism (CE-AFLP) screening

Amplified Fragment Length Polymorphism (AFLP) screening is a genome-wide genotyping strategy that has been widely used in plants and bacteria, but little has been reported concerning its use in humans. We investigated if the AFLP procedure could be coupled with high-throughput capillary electrophoresis (CE) for use in tumor diagnosis and classification. Using CE-AFLP, a series of molecular 'fingerprints' were generated for a set of gastric tumor and normal genomic DNA samples. The CE-AFLP procedure was qualitatively and quantitatively robust, and a variety of clustering tools were used to identify a specific DNA marker 'pattern' of 20 features that classified the tumor and normal samples to reasonable degrees of accuracy (Sensitivity 95%, Specificity 80%). The CE-AFLP-based approach also correctly classified 16 tumor samples, which in a previous study had exhibited no detectable genomic aberrations by comparative genome hybridization (CGH). This is the first reported application of CE-AFLP screening in tumor diagnosis. As the procedure is relatively inexpensive and requires minimal prior sequence knowledge and biological material, we suggest that CE-AFLP-based protocols may represent a promising new approach for DNA-based cancer screening and diagnosis.     

Sequence quality is maintained after multiple displacement amplification of non-invasively obtained macaque semen DNA

Whole genome amplification protocols are revolutionizing the fields of molecular and conservation biology as they open the possibility of obtaining a large number of copies of a complete genome from minute amounts of sample. Multiple displacement amplification (MDA) is a whole genome amplification technique based on the properties of the phi29 DNA polymerase, which leads to a uniform representation of the genome with very low error rates. In this study we performed MDA on 28 macaque DNA samples extracted from blood or non-invasively collected semen from which we obtained mitochondrial control region sequences both before and after MDA. The length of the readable sequences was longer for the original samples than for the MDA products, but the number of unresolved positions was comparable both before and after MDA. We conclude that the MDA technique is useful for increasing the amount of DNA for sequencing mitochondrial regions in the case of non-invasively collected semen samples.     

High-throughput S-SAP by fluorescent multiplex PCR and capillary electrophoresis in plants

The inherent replicative mode of transposition endows retrotransposons with considerable advantages as genetic tools in plant genome analysis. Here we present a high-throughput sequence-specific amplification polymorphism (S-SAP) method based on copia-like retrotransposons to fulfill the increasing desire of screening large numbers of samples in plants. Classic approach for digestion, ligation and pre-amplification was combined with optimized fluorescent multiplex PCR for simultaneously selective amplifying S-SAP fragments, and multiple S-SAPs were subsequently detected by capillary electrophoresis using ABI PRISM 3700 capillary instruments. Comparisons of results from multiplex PCR with simplex PCR, and from capillary electrophoresis with slab-gel electrophoresis demonstrated that this method is an efficient, economical, and accurate means for high-throughput and large-scale genotyping retrotransposon variation in plants.     

Reporting data from high-throughput screening of small-molecule libraries

Publications reporting results of small-molecule screens are becoming more common as academic researchers increasingly make use of high-throughput screening (HTS) facilities. However, no standards have been formally established for reporting small-molecule screening data, and often key information important for the evaluation and interpretation of results is omitted in published HTS protocols. Here, we propose concise guidelines for reporting small-molecule HTS data.     

灵芝麦角硫因高通量检测方法研究

麦角硫因(ergothioneine,EGT)基于其强抗氧化及体内消耗率低的特点,在人体内拥有多种重要的生理功能。但是在高产EGT微生物筛选和新菌株的选育研究中,现有的EGT检测方法因其步骤繁琐、使用的试剂和设备昂贵而亟待改进。本研究基于EGT的理化性质,建立了EGT-硫氰酸铁高通量快速检测体系,同时选用不同EGT产量的灵芝菌株以及灵芝融合新菌株对该检测体系的准确性进行验证。结果表明该方法可以快速准确地比较出样本间EGT产量高低,使原本需3~4天的工作缩短至2~3 h,HPLC验证结果显示,EGT-硫氰酸铁高通量快速检测体系效果良好,体系稳定。本研究结果将为高产EGT微生物的高通量筛选及高产EGT新菌株的选育提供新的方法和思路。     

High-throughgput phage-display screening in array format

Emerging technologies for the design and generation of human antibodies require improved approaches enabling their screening, characterization and validation. Currently, strategies based on ELISA or western blot are used to that aim. However, the ever increasing number of novel antibodies generated would benefit from the development of new high-throughput (HT) platforms facilitating rapid antibody identification and characterization. Herein, we describe a protein chip bearing recombinant phage particles and based on a large phage antibody library. In this paper we have set forth a novel implementation which provides a powerful and simple methodology enabling the identification of single-chain variable fragments (scFv). As a proof-of-principle of this method, we tested it with recombinant antigen (human recombinant interleukin 8). Additionally, we developed a novel bioinformatics tool that serves to compare this novel strategy with traditional methods. The method described here, together with associated informatics tools, is robust, relatively fast and represents a step-forward in protocols including phage library screenings.     

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.     

Phylogenetic network of the mtDNA haplogroup U in Northern Finland based on sequence analysis of the complete coding region by conformation-sensitive gel electrophoresis

Mutations in mtDNA have accumulated sequentially, and maternal lineages have diverged to form population-specific genotypes. Classification of the genotypes has been made based on differences found in restriction fragment analysis of the coding region or in the sequence of the hypervariable segment I. Both methods have shortcomings, as the former may not detect all the important polymorphisms and the latter makes use of a segment containing hypervariable nucleotide positions. Here, we have used conformation-sensitive gel electrophoresis (CSGE) to detect polymorphisms within the coding region of mtDNA from 22 Finns belonging to haplogroup U. Sixty-three overlapping PCR fragments covering the entire coding region were analyzed by CSGE, and the fragments that differed in their migration pattern were sequenced. CSGE proved to be a sensitive and specific method for identifying mtDNA substitutions. The phylogenetic network of the 22 coding-region sequences constituted a perfect tree, free of homoplasy, and provided several previously unidentified common polymorphisms characterizing subgroups of U. After contrasting this data with that of hypervariable segment I, we concluded that position 16192 seems to be prone to recurrent mutations and that position 16270 has experienced a back mutation. Interestingly, all 22 samples were found to belong to subcluster U5, suggesting that this subcluster is more frequent in Finns than in other European populations. Complete sequence data of the mtDNA yield a more reliable phylogenetic network and a more accurate classification of the haplogroups than previous ones. In medical genetics, such networks may help to decide between a rare polymorphism and a pathogenic mutation; in population genetics, the networks may enable more detailed analyses of population history and mtDNA evolution.     

Dynamic analysis of apoptosis using cyanine SYTO probes: From classical to microfluidic cytometry

Cell death is a stochastic process, often initiated and/or executed in a multi-pathway/multi-organelle fashion. Therefore, high-throughput single-cell analysis platforms are required to provide detailed characterization of kinetics and mechanisms of cell death in heterogeneous cell populations. However, there is still a largely unmet need for inert fluorescent probes, suitable for prolonged kinetic studies. Here, we compare the use of innovative adaptation of unsymmetrical SYTO dyes for dynamic real-time analysis of apoptosis in conventional as well as microfluidic chip-based systems. We show that cyanine SYTO probes allow non-invasive tracking of intracellular events over extended time. Easy handling and “stain-no wash” protocols open up new opportunities for high-throughput analysis and live-cell sorting. Furthermore, SYTO probes are easily adaptable for detection of cell death using automated microfluidic chip-based cytometry.Overall, the combined use of SYTO probes and state-of-the-art Lab-on-a-Chip platform emerges as a cost effective solution for automated drug screening compared to conventional Annexin V or TUNEL assays. In particular, it should allow for dynamic analysis of samples where low cell number has so far been an obstacle, e.g. primary cancer stems cells or circulating minimal residual tumors.     

High-throughput single-strand conformation polymorphism analysis by capillary electrophoresis

Mutation detection plays a great role in genetic and medical research and clinical diagnosis of inherited diseases and particular cancers. Single-strand conformation polymorphism (SSCP) analysis is one of the most popular methods for detection of mutations. Recently, automated capillary electrophoresis (CE) systems have been used in SSCP analysis instead of conventional slab gel electrophoresis. SSCP analysis in combination with CE is a rapid, simple, sensitive and high-throughput mutation screening tool, and has been successfully applied for mutation detection involving human tumor suppressor genes, oncogenes and disease-causing genes. The new technique has a great potential for mutation screening of large numbers of samples in clinical diagnosis. This review discusses basic issues about the methodology of SSCP analysis based on CE and summarizes several key applications.     

Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics.

Tissue microarray (TMA) technology provides a possibility to explore protein expression patterns in a multitude of normal and disease tissues in a high-throughput setting. Although TMAs have been used for analysis of tissue samples, robust methods for studying in vitro cultured cell lines and cell aspirates in a TMA format have been lacking. We have adopted a technique to homogeneously distribute cells in an agarose gel matrix, creating an artificial tissue. This enables simultaneous profiling of protein expression in suspension- and adherent-grown cell samples assembled in a microarray. In addition, the present study provides an optimized strategy for the basic laboratory steps to efficiently produce TMAs. Presented modifications resulted in an improved quality of specimens and a higher section yield compared with standard TMA production protocols. Sections from the generated cell TMAs were tested for immunohistochemical staining properties using 20 well-characterized antibodies. Comparison of immunoreactivity in cultured dispersed cells and corresponding cells in tissue samples showed congruent results for all tested antibodies. We conclude that a modified TMA technique, including cell samples, provides a valuable tool for high-throughput analysis of protein expression, and that this technique can be used for global approaches to explore the human proteome.     

High-Resolution Melt Analysis for Rapid Comparison of Bacterial Community Compositions

In the study of bacterial community composition, 16S rRNA gene amplicon sequencing is today among the preferred methods of analysis. The cost of nucleotide sequence analysis, including requisite computational and bioinformatic steps, however, takes up a large part of many research budgets. High-resolution melt (HRM) analysis is the study of the melt behavior of specific PCR products. Here we describe a novel high-throughput approach in which we used HRM analysis targeting the 16S rRNA gene to rapidly screen multiple complex samples for differences in bacterial community composition. We hypothesized that HRM analysis of amplified 16S rRNA genes from a soil ecosystem could be used as a screening tool to identify changes in bacterial community structure. This hypothesis was tested using a soil microcosm setup exposed to a total of six treatments representing different combinations of pesticide and fertilization treatments. The HRM analysis identified a shift in the bacterial community composition in two of the treatments, both including the soil fumigant Basamid GR. These results were confirmed with both denaturing gradient gel electrophoresis (DGGE) analysis and 454-based 16S rRNA gene amplicon sequencing. HRM analysis was shown to be a fast, high-throughput technique that can serve as an effective alternative to gel-based screening methods to monitor microbial community composition.