Semi-automated Protocol Research Articles

Abnormal patterns of cortical gyrification in velo-cardio-facial syndrome (deletion 22q11.2): An MRI study

Velo-cardio-facial syndrome (VCFS), also known as 22q11.2 deletion syndrome, is a common genetic condition associated with increased risk for developing schizophrenia. Given that cortical malformations play an integral role in the pattern of neuroanatomical alterations associated with VCFS, the aim of the present study was to quantify and localize gyral abnormalities. Magnetic resonance images were obtained on a 1.5 T scanner. The gyrification index (GI), a measure of the degree of cortical complexity, was differentially calculated for each lobe using a semi-automated protocol. The GI was calculated for 37 patients affected by VCFS as well as for 36 comparison individuals group-matched for age, handedness, and gender. The subjects affected by VCFS showed a significant decrease in the GI in the frontal and parietal lobes compared with the control group. The pattern of decreased gyrification in the frontal and parietal lobes further defines the structural changes associated with the syndrome and suggests underlying abnormalities in neural connectivity. Aberrant connectivity may be partially responsible for the cognitive and behavioral impairments in the syndrome, as well as the high incidence of schizophrenia among affected individuals.

A streamlined method for systematic, high resolution in situ analysis of mRNA distribution in plants

BackgroundIn situ hybridisation can provide cellular, and in some cases sub-cellular, resolution of mRNA levels within multicellular organisms and is widely used to provide spatial and temporal information on gene expression. However, standard protocols are complex and laborious to implement, restricting analysis to one or a few genes at any one time. Whole-mount and reverse transcriptase-PCR (RT-PCR) based protocols increase throughput, but can compromise both specificity and resolution. With the advent of genome-wide analysis of gene expression, there is an urgent need to develop high-throughput in situ methods that also provide high resolution.ResultsHere we describe the development of a method for performing high-throughput in situ hybridisations that retains both the high resolution and the specificity of the best manual versions. This refined semi-automated protocol has the potential for determining the spatial and temporal expression patterns of hundreds of genes in parallel on a variety of tissues. We show how tissue sections can be organized on microscope slides in a manner that allows the screening of multiple probes on each slide. Slide handling, hybridisation and processing steps have been streamlined providing a capacity of at least 200 probes per week (depending on the tissue type). The technique can be applied easily to different species and tissue types, and we illustrate this with wheat seed and Arabidopsis floral meristems, siliques and seedlings.ConclusionThe approach has the high specificity and high resolution of previous in situ methods while allowing for the analysis of several genes expression patterns in parallel. This method has the potential to provide an analysis of gene expression patterns at the genome level.

A semi‐automated digital microphotographic approach to measure meiofaunal biomass

Meiofauna studies often investigate community structure and function where biomass estimates and taxonomic analysis are required. Nondestructive methods of biomass estimation are necessary to preserve specimens for subsequent taxonomic analysis. A semi‐automated protocol to estimate meiofaunal biomass was developed to meet this need. The method improves upon previous indirect biomass techniques by using digital microphotography and analytical graphics software to obtain better estimates of biovolume. The technique is not fully automated because the digital images require some manipulation. Dry mass and carbon mass were estimated for two dominant components of marine benthic meiofauna (Nematoda and Harpacticoida) as the product of conversion factors and body volumes. The technique was validated by comparing indirect dry and carbon mass estimates to direct measurements using an analytical balance and carbon‐hydrogen‐nitrogen (CHN) elemental analyzer. No significant difference was found between the semi‐automated method and direct measurements for harpacticoid dry or carbon mass (P = 0.68 and P = 0.74, respectively), or nematode dry mass (P = 0.28) or carbon content (P = 0.17). The semi‐automated indirect method was used to estimate the biomass of meiofauna (13,279 harpacticoids and 12,288 nematodes) collected from the deep‐sea Gulf of Mexico. Estimated average wet mass was compared to direct analytical balance measurements from an earlier, independent study in the same area. Wet mass estimates generated by the indirect method (2.67 ± 0.86 µg/harpacticoid and 0.85 ± 4.78 µg/nematode) were similar to direct measurements in the earlier study (2.80 µg/harpacticoid and 0.85 µg/nematode). The semi‐automated indirect method is about three times faster than traditional microscope methods to measure body volume, estimates biomass comparably to direct methods, and conserves samples and images of samples for other analyses.

Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real-time PCR

With an increased emphasis on genotyping of single nucleotide polymorphisms (SNPs) in disease association studies, the genotyping platform of choice is constantly evolving. In addition, the development of more specific SNP assays and appropriate genotype validation applications is becoming increasingly critical to elucidate ambiguous genotypes. In this study, we have used SNP specific Locked Nucleic Acid (LNA) hybridization probes on a real-time PCR platform to genotype an association cohort and propose three criteria to address ambiguous genotypes. Based on the kinetic properties of PCR amplification, the three criteria address PCR amplification efficiency, the net fluorescent difference between maximal and minimal fluorescent signals and the beginning of the exponential growth phase of the reaction. Initially observed SNP allelic discrimination curves were confirmed by DNA sequencing (n = 50) and application of our three genotype criteria corroborated both sequencing and observed real-time PCR results. In addition, the tested Caucasian association cohort was in Hardy-Weinberg equilibrium and observed allele frequencies were very similar to two independently tested Caucasian association cohorts for the same tested SNP. We present here a novel approach to effectively determine ambiguous genotypes generated from a real-time PCR platform. Application of our three novel criteria provides an easy to use semi-automated genotype confirmation protocol.

In-Situ Measurements of Low-Level Mercury Vapor Exposure from Dental Amalgam with Zeeman Atomic Absorption Spectroscopy

Alongside food, emissions from amalgam fillings are an essential contribution to man's mercury burden. Previous methods for the determination of intraoral mercury vapor (Hg°) release used principally some form of preconcentration of Hg on gold (film or wool), allowing relatively few measurements with unknown precision and sensitivity at selected times. Recently available computer-controlled Hg detectors operating on Zeeman atomic absorption spectroscopy (ZAAS) facilitate the direct real-time measurement of Hg° concentrations. It was the aim to adapt this method for a comparative investigation of emission processes from fillings in situ and from amalgam specimens in vitro. In addition to the ZAAS instrument, the apparatus consisted of a pump, magnetic valves, an electronic flow controller and a handle with a disposable mouth piece for aspiration of oral air. A programmable timer integrated the computer-controlled instrument operation and the data collection into a standard sampling protocol. A fast exponential decay of the emission was found after stimulation of amalgam specimens and of fillings in situ (halftimes 8.6 and 10.7 min). Precision was evaluated by a series of measurements on a single patient which indicated a consistently low coefficient of variation between 18% and 25%. After insertion of a few new fillings, sensitivity was high enough to detect a significant increase in emission against the background emission from the majority of old fillings. Zeeman-AAS in connection with a semi-automated sampling protocol and data storage provides precise in-situ measurements of Hg° emission from dental amalgam with real-time resolution. This facilitates the detailed exploration of the Hg° release kinetics and the applicability to large-scale studies.

Analysis of sequence variations in several human genes using phosphoramidite bond DNA fragmentation and chip-based MALDI-TOF.

The challenge in the postgenome era is to measure sequence variations over large genomic regions in numerous patient samples. This massive amount of work can only be completed if more accurate, cost-effective, and high-throughput solutions become available. Here we describe a novel DNA fragmentation approach for single nucleotide polymorphism (SNP) discovery and sequence validation. The base-specific cleavage is achieved by creating primer extension products, in which acid-labile phosphoramidite (P-N) bonds replace the 5' phosphodiester bonds of newly incorporated pyrimidine nucleotides. Sequence variations are detected by hydrolysis of this acid-labile bond and MALDI-TOF analysis of the resulting fragments. In this study, we developed a robust protocol for P-N-bond fragmentation and investigated additional ways to improve its sensitivity and reproducibility. We also present the analysis of several human genomic targets ranging from 100-450 bp in length. By using a semiautomated sample processing protocol, we investigated an array of SNPs within a 240-bp segment of the NFKBIA gene in 48 human DNA samples. We identified and measured frequencies for the two common SNPs in the 3'UTR of NFKBIA (separated by 123 bp) and then confirmed these values in an independent genotyping experiment. The calculated allele frequencies in white and African American groups differed significantly, yet both fit Hardy-Weinberg expectations. This demonstrates the utility and effectiveness of PN-bond DNA fragmentation and subsequent MALDI-TOF MS analysis for the high-throughput discovery and measurement of sequence variations in fragments up to 0.5 kb in length in multiple human blood DNA samples.

Effects of Domain Dissection on the Folding and Stability of the 43 kDa Protein PGK Probed by NMR

The characterization of early folding intermediates is key to understanding the protein folding process. Previous studies of the N-domain of phosphoglycerate kinase (PGK) from Bacillus stearothermophilus combined equilibrium amide exchange data with a kinetic model derived from stopped-flow kinetics. Together, these implied the rapid formation of an intermediate with extensive native-like hydrogen bonding. However, there was an absence of protection in the region proximal to the C-domain in the intact protein. We now report data for the intact PGK molecule, which at 394 residues constitutes a major extension to the protein size for which such data can be acquired. The methods utilised to achieve the backbone assignment are described in detail, including a semi-automated protocol based on a simulated annealing Monte Carlo technique. A substantial increase in the stability of the contact region is observed, allowing protection to be inferred on both faces of the β-sheet in the intermediate. Thus, the entire N-domain acts concertedly in the formation of the kinetic refolding intermediate rather than there existing a distinct local folding nucleus.

BioMagResBank database with sets of experimental NMR constraints corresponding to the structures of over 1400 biomolecules deposited in the Protein Data Bank.

Experimental constraints associated with NMR structures are available from the Protein Data Bank (PDB) in the form of "Magnetic Resonance" (MR) files. These files contain multiple types of data concatenated without boundary markers and are difficult to use for further research. Reported here are the results of a project initiated to annotate, archive, and disseminate these data to the research community from a searchable resource in a uniform format. The MR files from a set of 1410 NMR structures were analyzed and their original constituent data blocks annotated as to data type using a semi-automated protocol. A new software program called Wattos was then used to parse and archive the data in a relational database. From the total number of MR file blocks annotated as constraints, it proved possible to parse 84% (3337/3975). The constraint lists that were parsed correspond to three data types (2511 distance, 788 dihedral angle, and 38 residual dipolar couplings lists) from the three most popular software packages used in NMR structure determination: XPLOR/CNS (2520 lists), DISCOVER (412 lists), and DYANA/DIANA (405 lists). These constraints were then mapped to a developmental version of the BioMagResBank (BMRB) data model. A total of 31 data types originating from 16 programs have been classified, with the NOE distance constraint being the most commonly observed. The results serve as a model for the development of standards for NMR constraint deposition in computer-readable form. The constraints are updated regularly and are available from the BMRB web site (http://www.bmrb.wisc.edu).

Genomic semi-automated cycle sequencing as a sensitive screening technique for p53 mutations in frozen tumor samples.

Single-strand conformational polymorphism (SSCP) is the most widely used method for p53 gene mutation screening. Nucleotide sequence analysis is considered more sensitive for detection of mutations. We established a genomic semi-automated cycle sequencing protocol suitable for p53 gene mutation screening. The technique was applied to 44 SSCP-negative frozen ovarian cancer samples: Eleven mutations (11/44, 25%) were found, 6 point missense mutations, 3 silent point mutations, 1 nonsense mutation and 1 single-base deletion. Heterozygous mutations were readily detectable. Genomic semi-automated cycle sequencing is a sensitive, time-effective screening method requiring only small amounts of tumor tissue.

Quantitation of satellite cell proliferation in vivo using image analysis.

A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei+satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 +/- 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 +/- 462 microns3 of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.

A robotics-assisted procedure for large scale cystic fibrosis mutation analysis.

We describe a convenient, efficient, semiautomated protocol for assaying large numbers of DNA samples for over 20 mutations causing cystic fibrosis. The protocol uses the following: (1) a programmable robotic workstation to perform rapid pipetting and dot-blotting operations, (2) an allele-specific oligonucleotide hybridization in a single water bath without correcting for G+C content of oligonucleotides, and (3) a combinatorial system that allows direct determination of the genotype for more frequent mutations. We have used this system routinely for 16 months for carrier detection and for diagnosis of cystic fibrosis. The method can be readily applied to any combination of allele-specific oligonucleotide assays whether for multiple alleles at one locus or for a few alleles at multiple loci.

Autoimmune Polyglandular Disease Type I

The pathogenetic background of human autoimmunity is only partially understood. By discovering the defective gene causing the autosomal recessive polyglandular autoimmune disease type I (PGD I, APECED) we hope to provide new insights into autoimmune responses in general. Here we have taken advantage of newly developed amplifiable multiallelic microsatellite markers and performed the analyses using the microtiter well format of the polymerase chain reaction. This rapid semiautomated protocol was applied to analyze 62 assigned highly polymorphic loci. The linkage analyses coupled with the EXCLUDE analysis resulted in an exclusion map of this polyglandular autoimmune disease and in the preliminary assignment of the APECED locus to chromosome 22. The method proved to be an effective and economical tool for gene mapping compared with standard blotting and hybridization.