Utility of mitochondrial CO1 sequences for species discrimination of Spirotrichea ciliates (Protozoa, Ciliophora).

Ciliates are a diverse species group of the Protozoa, and nuclear and mitochondrial genes have been utilized to discover new species and discriminate closely related species. The mitochondrial cytochrome c oxidase subunit 1 (CO1) gene has been used to discriminate metazoan species and has also been applied for some groups in the phylum Ciliophora. However, it is difficult to produce a universal primer as a standard barcode, because unlike metazoans, mitochondrial DNA sequences of ciliates are long and highly variable. Therefore, to design the new primer set, we sequenced the mitochondrial genomes of two pseudokeronopsids in the class Spirotrichea using next-generation sequencing technology (HiSeq™ 2000). Based on putative CO1 gene fragments of the pseudokeronopsids, we designed the new primer set and successfully sequenced the CO1 of 69 populations representing 47 species (five orders, 14 families, and 27 genera). We found that CO1 showed higher resolution for separating congeneric species than did nuclear SSU rRNA gene sequences, and we identified some putative cryptic species.

The Human OligoGenome Resource: a database of oligonucleotide capture probes for resequencing target regions across the human genome.

Recent exponential growth in the throughput of next-generation DNA sequencing platforms has dramatically spurred the use of accessible and scalable targeted resequencing approaches. This includes candidate region diagnostic resequencing and novel variant validation from whole genome or exome sequencing analysis. We have previously demonstrated that selective genomic circularization is a robust in-solution approach for capturing and resequencing thousands of target human genome loci such as exons and regulatory sequences. To facilitate the design and production of customized capture assays for any given region in the human genome, we developed the Human OligoGenome Resource (http://oligogenome.stanford.edu/). This online database contains over 21 million capture oligonucleotide sequences. It enables one to create customized and highly multiplexed resequencing assays of target regions across the human genome and is not restricted to coding regions. In total, this resource provides 92.1% in silico coverage of the human genome. The online server allows researchers to download a complete repository of oligonucleotide probes and design customized capture assays to target multiple regions throughout the human genome. The website has query tools for selecting and evaluating capture oligonucleotides from specified genomic regions.

Development of a common oligonucleotide reference standard for microarray data normalization and comparison across different microbial communities.

High-density functional gene arrays have become a powerful tool for environmental microbial detection and characterization. However, microarray data normalization and comparison for this type of microarray remain a challenge in environmental microbiology studies because some commonly used normalization methods (e.g., genomic DNA) for the study of pure cultures are not applicable. In this study, we developed a common oligonucleotide reference standard (CORS) method to address this problem. A unique 50-mer reference oligonucleotide probe was selected to co-spot with gene probes for each array feature. The complementary sequence was synthesized and labeled for use as the reference target, which was then spiked and cohybridized with each sample. The signal intensity of this reference target was used for microarray data normalization and comparison. The optimal amount or concentration were determined to be ca. 0.5 to 2.5% of a gene probe for the reference probe and ca. 0.25 to 1.25 fmol/microl for the reference target based on our evaluation with a pilot array. The CORS method was then compared to dye swap and genomic DNA normalization methods using the Desulfovibrio vulgaris whole-genome microarray, and significant linear correlations were observed. This method was then applied to a functional gene array to analyze soil microbial communities, and the results demonstrated that the variation of signal intensities among replicates based on the CORS method was significantly lower than the total intensity normalization method. The developed CORS provides a useful approach for microarray data normalization and comparison for studies of complex microbial communities.

Specific detection of bacterial pathogens using oligonucleotide microarrays generated from hydrolysis PCR probe sequences.

A move away from high-density screening array formats and the implementation of probes specifically identifying targets for application in low-density hybridization capture analyses is growing in importance. Some of the highest specificity for bioassays that encompasses use of hybridization has enlisted hydrolysis probes in real-time PCR. It is demonstrated that by employing 5'-end-tethering to glass of hydrolysis PCR probe sequences that these can be employed as capture probe sequences. The probes retain hybridization binding specificity to their PCR amplicons and specificity of hybridization was achieved across all probes at equivalent stringency. This suggests that probes identified for use in hydrolysis PCR using one set of temperature cycling parameter can also be applied to a microarray, where again the probes all exhibit similar specificity of interact at analogous hybridization conditions. The procedure permits the exact same PCR amplicon sequences to be used in both quantitative PCR and qualitative microarray assay formats.

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.

Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays.

BACKGROUND: Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. RESULTS: Significantly (p < 0.01) improved DNA retention was achieved printing in 15% DMSO/1.5 M betaine compared to the vendor recommended buffers. Introduction of tracking oligonucleotide did not effect hybridization efficiency or introduce ratio measurement bias in hybridizations between M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p < 0.05) and there were no significant differences in Pearson's correlation coefficients of ratio data between replicates possessing (0.72 +/- 0.07), replicates lacking (0.74 +/- 0.10), or replicates with and without (0.70 +/- 0.04) the tracking oligonucleotide. ANOVA analysis confirmed the tracking oligonucleotide introduced no bias. Titrating target-specific oligonucleotide (40 microM to 0.78 microM) in the presence of 0.5 microM tracking oligonucleotide, revealed a fluorescein fluorescence inversely related to target-specific oligonucleotide molarity, making tracking oligonucleotide signal useful for quality control measurements and differentiating false negatives (synthesis failures and mechanical misses) from true negatives (no gene expression). CONCLUSIONS: This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

Avoidance of nonspecific hybridization by employing oligonucleotide micro-arrays generated from hydrolysis polymerase chain reaction probe sequences.

We have developed a low-density oligonucleotide-based micro-array where 5'-end-tethered capture probe sequences were derived from Primer Express software. The capture probes represent hydrolysis probe sequences devoid of any fluorochromes and were shown to retain hybridization binding specificity to their amplicons; hybridization specificity was retained independent to probe sequences. This procedure allowed the specificity of each capture probe to be verified using real-time polymerase chain reaction (PCR) in the presence of nucleic acid sequences typically expected to be present within a sample and therefore has reduced possibility of nonspecific hybridization when used in a micro-array format. We propose that specificity-validated probes are applied to form a micro-array for the purpose of general target screening, with incumbent multiparallelization and cost and time savings. However, if required, the subset of probe sequences of interest can be used for quantitative assessment in conventional real-time PCR.

Size and composition of T-cell receptor delta (TCRD) junctional sequences are not predictive of the sensitivity of clonospecific oligonucleotides designed for detection of minimal residual disease in acute lymphoblastic leukemia.

We characterized 168 junctional regions of T-cell receptor delta (TCRD) rearrangements from 116 children with acute lymphoblastic leukemia (ALL) (101 with precursor B-cell ALL, 15 with T-cell ALL). Application of 101 allele-specific oligonucleotide (ASO) probes representing 85 Vdelta2Ddelta3, 10 Ddelta2Ddelta3, 3 Vdelta1Jdelta1, 1 Vdelta3Jdelta1, and 2 Ddelta2Jdelta1 junctions for the detection of minimal residual disease (MRD) revealed detection levels of 10(-4) to 10(-6) leukemia cells in the vast majority of cases (93 of 101). Of interest was that neither the N, D, P (nontemplated, diversity, palindromic) content and length of the junctional regions nor the number of nucleotides deleted from the flanking V, D, or J (variable, diversity, joining) elements correlated with the sensitivity of ASO probes. These data indicated that in ALL TCRD rearrangements can serve as suitable tools for the detection of MRD irrespective of the specific composition of the junctional region.

Short, 12 mer fluorescently labeled methylphosphonated oligonucleotides to visualize beta-actin MRNA in vivo.

A pair of fluorescently labeled antisense ( complementary to beta-actin mRNA) or control methylphosphonated DNA 12-mers were introduced into live cells. After fixation their distribution throughout the cell was compared to the localization pattern for the pair of control oligos.The distribution of the two sets of oligos differed in that there was a distinct pool of antisense probes that were detected at elevated levels in the leading edge of fibroblast and cortical underlining. The resulting fluorescence patterns of antisense probes colocalized and were analogous to labeling pattern already described and produced by in situ hybridization. The length of each of the probe destabilized binding to mismatched sequences at physiological temperature, while the overall length of the pair gave a unique, highly sequence specific recognition of a target sequence. Simultaneous, in vivo application of multiple probes let include internal controls into the experimental setup, in order to distinguish different distributions of antisense and control probes in the same specimen.

Evaluation of new linkers and synthetic methods for internal modified oligonucleotides.

Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine. The products were compared in functional assays. They performed very similarly both in a fluorescence-based melting point assay as well as in quantitative PCR. Another set of beta-actin probes were synthesized utilizing N4 [N-2-(ethylene glycol ethyl)-5-methyl]cytidine and solid-phase Tamra amidite addition at positions flanking those of the uridine. These versions gave lower T(m)s than either uridine-labeled probe and did not work as well in quantitative PCR. A control experiment using oligonucleotides with the same modified residues but without fluorophores attached revealed the same trend as the T(m) study of internal Tamra-labeled probes. Experimental details for the synthesis, purification, and testing are presented.

A simple method to improve probe set estimates from oligonucleotide arrays.

A popular commercially available oligonucleotide microarray technology employs sets of 25 base pair oligonucleotide probes for measurement of gene expression levels. A mathematical algorithm is required to compute an estimate of gene expression from the multiple probes. Previously proposed methods for summarizing gene expression data have either been substantially ad hoc or have relied on model assumptions that may be easily violated. Here we present a new algorithm for calculating gene expression from probe sets. Our approach is functionally related to leave-one-out cross-validation, a non-parametric statistical technique that is often applied in limited data situations. We illustrate this approach using data from our study seeking a molecular fingerprint of STAT3 regulated genes for early detection of human cancer.

A rapid method for semiquantitative analysis of the human V beta-repertoire using TaqManR PCR.

Analysis of the V beta-repertoire of antigen-reactive T cell populations can be approached using either flow-cytometry or PCR-based techniques. While the former method requires a complete set of V beta-specific monoclonal antibodies (mAbs) and large cell numbers for analysis, the latter is both time-consuming and labour-intensive. To circumvent the drawbacks of both these methods we have employed the recently developed technique of TaqManR PCR to analyse the V beta-usage of human T cell populations. TaqManR PCR is based on the 5'-->3' nuclease activity of Taq polymerase. During PCR amplification an internal oligonucleotide probe, that is labelled with a fluorescent reporter and a quencher dye, is cleaved by Taq polymerase. After cleavage, quenching of the reporter dye is lost and reporter fluorescence can be detected with a fluorescence plate reader. Using one C beta-specific fluorogenic probe and a panel of V beta-specific primers, we show that fluorescence-detected amplification of TCR beta cDNA is V beta-specific and linear within a 2-3-log range of template concentration. The sensitivity of TaqManR PCR is comparable to conventional detection of PCR-products by agarose gel staining, while processing time is reduced. Furthermore, superantigen-induced skewing of the V beta-repertoire of human T cells is readily detected with this method. Thus TaqManR PCR is a reliable and fast method for semiquantitative analysis of the V beta-repertoire of human T cell populations.