Accuracy of genotyping for single nucleotide polymorphisms by a microarray-based single nucleotide polymorphism typing method involving hybridization of short allele-specific oligonucleotides.

Advances in technologies for identifying genetic polymorphisms rapidly and accurately will dramatically accelerate the discovery of disease-related genes. Among a variety of newly described methods for rapid typing of single-nucleotide polymorphisms (SNPs), gene detection using DNA microarrays is gradually achieving widespread use. This method involves the use of short (11- to 13-mer) allele-specific oligonucleotides. This method allows simultaneous analysis of many SNPs in DNAs from a large number of individuals, in a single experiment. In this work, we evaluated the accuracy of a new microarray-based short allele-specific oligonucleotide (ASO) hybridization method. There is a 96-well formatted array on a single plate, in which up to 256 spots are included in each well. Fluorescent probes for our experiments were produced by multiplex PCR amplification often target SNP-containing regions. We genotyped 192 individuals across a panel of ten single base variations, which included an insertion/deletion polymorphism. For comparison, we genotyped the same individuals for the same SNPs by the method of single-base extension with fluorescence detection. The typing accuracies of the microarray-based PCR-ASO and single-base extension methods were calculated as 99.9% and 99.1%, respectively, on the basis of genotyping results determined by direct sequencing. We conclude that the microarray-based hybridization method using short ASO probes represents a potential breakthrough technology for typing large numbers of SNPs rapidly and efficiently.     

Allelic dosage analysis with genotyping microarrays

Genomic alternations, including dosage and allelic imbalance, constitute a major basis of neoplastic and other genetic disorders. Using oligonucleotide genotyping microarrays, here we report the development and usage of an algorithm, called genome imbalance map (GIM) algorithm, for allelic as well as total gene dosage analysis. Using the GIM algorithm, global genome imbalance status at over 100,000 loci was simultaneously analyzed with unprecedented accuracy and allelic discrimination.     

Accessing single nucleotide polymorphisms in genomic DNA by direct multiplex polymerase chain reaction amplification on oligonucleotide microarrays

This study introduces a DNA microarray-based genotyping system for accessing single nucleotide polymorphisms (SNPs) directly from a genomic DNA sample. The described one-step approach combines multiplex amplification and allele-specific solid-phase PCR into an on-chip reaction platform. The multiplex amplification of genomic DNA and the genotyping reaction are both performed directly on the microarray in a single reaction. Oligonucleotides that interrogate single nucleotide positions within multiple genomic regions of interest are covalently tethered to a glass chip, allowing quick analysis of reaction products by fluorescence scanning. Due to a fourfold SNP detection approach employing simultaneous probing of sense and antisense strand information, genotypes can be automatically assigned and validated using a simple computer algorithm. We used the described procedure for parallel genotyping of 10 different polymorphisms in a single reaction and successfully analyzed more than 100 human DNA samples. More than 99% of genotype data were in agreement with data obtained in control experiments with allele-specific oligonucleotide hybridization and capillary sequencing. Our results suggest that this approach might constitute a powerful tool for the analysis of genetic variation.     

Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Gene-set analysis and reduction

Gene-set analysis aims to identify differentially expressedgene sets (pathways) by a phenotype in DNA microarray studies.We review here important methodological aspects of gene-setanalysis and illustrate them with varying performance of severalmethods proposed in the literature. We emphasize the importanceof distinguishing between ‘self-contained’ versus‘competitive’ methods, following Goeman and Bühlmann.We also discuss reducing a gene set to its subset, consistingof ‘core members’ that chiefly contribute to thestatistical significance of the differential expression of theinitial gene set by phenotype. Significance analysis of microarrayfor gene-set reduction (SAM-GSR) can be used for an analyticalreduction of gene sets to their core subsets. We apply SAM-GSRon a microarray dataset for identifying biological gene sets(pathways) whose gene expressions are associated with p53 mutationin cancer cell lines. Codes to implement SAM-GSR in the statisticalpackage R can be downloaded from http://www.ualberta.ca/~yyasui/homepage.html.      

Microarray analysis of genes differentially expressed in hepG2 cells cultured in simulated microgravity: Preliminary report

Developed at NASA, the rotary cell culture system (RCCS) allows the creation of unique microgravity environment of low shear force, high-mass transfer, and enables three-dimensional (3D) cell culture of dissimilar cell types. Recently we demonstrated that a simulated microgravity is conducive for maintaining long-term cultures of functional hepatocytes and promote 3D cell assembly. Using deoxyribonucleic acid (DNA) microarray technology, it is now possible to measure the levels of thousands of different messenger ribonucleic acids (mRNAs) in a single hybridization step. This technique is particularly powerful for comparing gene expression in the same tissue under different environmental conditions. The aim of this research was to analyze gene expression of hepatoblastoma cell line (HepG2) during early stage of 3D-cell assembly in simulated microgravity. For this, mRNA from HepG2 cultured in the RCCS was analyzed by deoxyribonucleic acid microarray. Analyses of HepG2 mRNA by using 6K glass DNA microarray revealed changes in expression of 95 genes (overexpression of 85 genes and downregulation of 10 genes). Our preliminary results indicated that simulated microgravity modifies the expression of several genes and that microarray technology may provide new understanding of the fundamental biological questions of how gravity affects the development and function of individual cells.     

Estimation of relative allele frequencies of single-nucleotide polymorphisms in different populations by microarray hybridization of pooled DNA

Single-nucleotide polymorphisms (SNPs) are considered useful polymorphic markers for genetic studies of polygenic traits. A new practical approach to high-throughput genotyping of SNPs in a large number of individuals is needed in association study and other studies on relationships between genes and diseases. We have developed an accurate and high-throughput method for determining the allele frequencies by pooling the DNA samples and applying a DNA microarray hybridization analysis. In this method, the combination of the microarray, DNA pooling, probe pair hybridization, and fluorescent ratio analysis solves the dual problems of parallel multiple sample analysis, and parallel multiplex SNP genotyping for association study. Multiple DNA samples are immobilized on a slide and a single hybridization is performed with a pool of allele-specific oligonucleotide probes. The results of this study show that hybridization of microarray from pooled DNA samples can accurately obtain estimates of absolute allele frequencies in a sample pool. This method can also be used to identify differences in allele frequencies in distinct populations. It is amenable to automation and is suitable for immediate utilization for high-throughput genotyping of SNP.     

D-Ribulose Production by a Thiamine-requiring Corynebacterium Species

The effect of thiamine on the D-ribulose production from gluconate by a thiamine-requiring Corynebacterium species was investigated. The D-ribulose production by the cells previously grown in a thiamine-deficient medium was higher than that by the cells grown in a thiamine-rich medium and supplementation of the thiamine-deficient cells with thiamine resulted in a significant depression of the D-ribulose production. Gluconokinase and NADP-linked phosphogluconate dehydrogenase were detected in the cell-free extract of this organism. Oxidation and anaerobic dissimilation of D-ribose 5-phosphate by the cell-free extract of the thiamine-deficient cells are reduced and the addition of thiamine pyrophosphate to the extract enhanced the catabolic activities for D-ribose 5-phosphate. These results suggest that the accumulation of D-ribulose by the thiamine-deficient cells is a consequence of a reduction of transketolase activity.     

Tissue-specific gene expression in soybean (Glycine max) detected by cDNA microarray analysis

We have constructed cDNA microarrays for soybean (Glycine maxL. Merrill), containing approximately 4,100 Unigene ESTs derived from axenic roots, to evaluate their application and utility for functional genomics of organ differentiation in legumes. We assessed microarray technology by conducting studies to evaluate the accuracy of microarray data and have found them to be both reliable and reproducible in repeat hybridisations. Several ESTs showed high levels (50 fold) of differential expression in either root or shoot tissue of soybean. A small number of physiologically interesting, and differentially expressed sequences found by microarray analysis were verified by both quantitative real-time RT-PCR and Northern blot analysis. There was a linear correlation (r² = 0.99, over 5 orders of magnitude) between microarray and quantitative real-time RT-PCR data. Microarray analysis of soybean has enormous potential not only for the discovery of new genes involved in tissue differentiation and function, but also to study the expression of previously characterised genes, gene networks and gene interactions in wild-type, mutant or transgenic plants.     

Electrochemical biosensor based on base-stacking-dependent DNA hybridization assay for protein detection

An antibody-based electrochemical biosensing platform has been developed and used for the detection of protein. In the presence of the target, an antibody pair binds to the protein simultaneously, which causes two oligo-DNAs conjugated with the antibody pair to hybridize to each other and become a big “stem–loop” structure. Subsequently, the longer oligo-DNA of the stem, with a methylene blue (MB) label at the terminal, hybridizes stably with capture DNA owing to the enhancement of base stacking. The strong redox current signal of MB is used for protein quantification. Using α-fetoprotein (AFP) as a model, the proposed method could detect AFP at a concentration as low as 2 pg ml⁻¹ with a dynamic range of 4 orders of magnitude, which approaches traditional assays such as enzyme-linked immunosorbent assay.     

Monitoring the gene expression profiles of doxorubicin-resistant acute myelocytic leukemia cells by DNA microarray analysis

Acquired drug-resistance phenotype is a key factor in the relapse of patients suffering hematological malignancies. In order to investigate the genes involved in drug resistance, a human leukemia cell line that is resistant to doxorubicin, an anthracycline anticancer agent (AML-2/DX100), was selected and its gene expression profile was analyzed using a cDNA microarray. A number of genes were differentially expressed in the AML-2/DX100 cells, compared with the wild type (AML-2/WT). Pro-apoptotic genes such as TNFSF7 and p21 (Cip1/Waf1) were significantly down-regulated, whereas the IKBKB, PCNA, stathmin 1, MCM5, MMP-2 and MRP1 genes, which are involved in anti-apoptotic or cell cycle progression, were over-expressed. The AML-2/DX100 cells were also resistant to other anticancer drugs, including daunorubicin and camptothecin, and the expression levels of the differentially regulated genes such as STMN1, MMP-2 and CTSG, were constantly maintained. This suggests that the deregulated genes obtained from the DNA microarray analysis in a cell line model of drug resistance might contribute to the acquired drug resistance after chronic exposure.     

Differentiation of Coxiella burnetii isolates by sequence determination and PCR-restriction fragment length polymorphism analysis of isocitrate dehydrogenase gene

The isocitrate dehydrogenase (icd) gene of Coxiella burnetii was cloned and sequenced to differentiate between isolates with various geographic origins and phenotypic properties. Based on the gene sequences all 19 isolates studied could be divided into three groups. Group 1 contained isolates originating from acute cases of Q fever, ticks and cows. Groups 2 and 3 included isolates from chronic Q fever patients and a prototype strain from an aborted goat. Although the icd gene profiles were different among isolates of the latter two groups, there were two base differences common for both groups which could be used as markers to distinguish them from group 1 isolates. Based on one of the markers a simple method using PCR-restriction fragment length polymorphism analysis was developed for rapid differentiation of C. burnetii isolates as well as for direct detection and differentiation of the bacterium in human serum samples. Taken together, the study results suggest that the icd-based differentiation method may be useful in clinical investigation of Coxiella infections.     

Correlation analysis of external RNA controls reveals its utility for assessment of microarray assay

Quality control of a microarray experiment has become an important issue for both research and regulation. External RNA controls (ERCs), which can be either added to the total RNA level (tERCs) or introduced right before hybridization (cERCs), are designed and recommended by commercial microarray platforms for assessment of performance of a microarray experiment. However, the utility of ERCs has not been fully realized mainly due to the lack of sufficient data resources. The US Food and Drug Administration (FDA)-led community-wide Microarray Quality Control (MAQC) study generates a large amount of microarray data with implementation of ERCs across several commercial microarray platforms. The utility of ERCs in quality control by assessing the ERCs’ concentration-response behavior was investigated in the MAQC study. In this work, an ERC-based correlation analysis was conducted to assess the quality of a microarray experiment. We found that the pairwise correlations of tERCs are sample independent, indicating that the array data obtained from different biological samples can be treated as technical replicates in analysis of tERCs. Consequently, the commonly used quality control method of applying correlation analysis on technical replicates can be adopted for assessing array performance based on different biological samples using tERCs. The proposed approach is sensitive to identifying outlying assays and is not dependent on the choice of normalization method.     

A single nucleotide polymorphism melt curve assay employing an intercalating dye probe fluorescence resonance energy transfer for forensic analysis

The characterization and use of DNA sequence polymorphisms are an important aspect of forensic analysis. A number of approaches are being explored for single nucleotide polymorphism (SNP) genotyping, but current detection methods are subject to limitations that adversely impact their utility for forensic analysis. We have developed a novel method for genotyping both single and multiple SNPs that uses an intercalating dye and a probe labeled with a single fluorophore to affect a fluorescence energy transfer. Melting curve analysis is then used to distinguish true alleles from mismatched alleles. We term the new method dye probe fluorescence resonance energy transfer (dpFRET). In the current work, development proceeded at first with synthetic DNA template testing to establish proof of concept for the chemistry involved, followed by the design of polymerase chain reaction (PCR)-based genomic DNA assays to demonstrate potential forensic applications. The loci chosen for testing included both nuclear (MHC DRB) and mitochondrial DNA (cytochrome b) genes. A preliminary assessment of the sensitivity limits of the technology indicated that dpFRET was capable of accurately genotyping DNA from one single diploid cell equivalent. This technology could also potentially impact a wide range of nonforensic disciplines to aid in discovery, screening, and association of DNA sequence polymorphisms.     

Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis

DNA analysis is an important technology with respect to diagnosis of infectious disease and tailored medication. In this study, we developed a novel bioluminescent assay for pyrophosphate, and it was applied to single-nucleotide polymorphism (SNP) analysis using one-base extension reaction. The principle of this method is as follows. A specific primer within each aliquot possessing a short 3′ end of the base of interest was hybridized to the single-stranded DNA template. Subsequently, (exo-)Klenow DNA polymerase and one of either α-thio-dATP, dTTP, dGTP, or dCTP were added and incubated for 1 min. Pyrophosphate released by DNA polymerase is converted to ATP by pyruvate phosphate dikinase (PPDK), and the concentration of ATP is determined using the firefly luciferase reaction. This method, which does not require expensive equipment, can be used to rapidly monitor one point mutation in the gene.     

Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay

The frequent occurrence of harmful algal blooms (HABs) is a pressing topic in marine research. An integrated sandwich hybridization and nuclease protection assay was established to qualitatively and quantitatively detect 12 harmful algal species. This method demonstrated good reliability, specificity and accuracy for analyzing samples from individual and mixed cultures, as well as field collection, and cell volumes were positively correlated to the slopes of calibration curves. The lowest quantitative detection limits were those concentrations observed during blooms; thus, this technique provides an efficient alternative to microscopy for rapid identification and quantitation of harmful algal species and could be routinely used to monitor phytoplankton in field surveys.     

Mutation analysis using the restriction site mutation (RSM) assay

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109–121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117–123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl-N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.