Oligonucleotide Probe Arrays Research Articles

Multiple Bacteria Identification in the Point-of-Care: an Old Method Serving a New Approach.

The accurate diagnosis of bacterial infections is of critical importance for effective treatment decisions. Due to the multietiologic nature of most infectious diseases, multiplex assays are essential for diagnostics. However, multiplexability in nucleic acid amplification-based methods commonly resorts to multiple primers and/or multiple reaction chambers, which increases analysis cost and complexity. Herein, a polymerase chain reaction (PCR) offer method based on a universal pair of primers and an array of specific oligonucleotide probes was developed through the analysis of the bacterial 16S ribosomal RNA gene. The detection system consisted of DNA hybridization over an array of magnetoresistive sensors in a microfabricated biochip coupled to an electronic reader. Immobilized probes interrogated single-stranded biotinylated amplicons and were obtained using asymmetric PCR. Moreover, they were magnetically labelled with streptavidin-coated superparamagnetic nanoparticles. The benchmarking of the system was demonstrated to detect five major bovine mastitis-causing pathogens: Escherichia coli, Klebsiella sp., Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae. All selected probes proved to specifically detect their respective amplicon without significant cross reactivity. A calibration curve was performed for S. agalactiae, which demonstrates demonstrating a limit of detection below 30 fg/µL. Thus, a sensitive and specific multiplex detection assay was established, demonstrating its potential as a bioanalytical device for point-of-care applications.

Human leukocyte antigen typing using a knowledge base coupled with a high-throughput oligonucleotide probe array analysis.

Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world’s populations, benefiting both global public health and personalized health care.

The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation.

The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development.

Gold Nanoparticle Assists SNP Detection at Room Temperature in the NanoBioArray Chip

This study employs a NanoBioArray (NBA) chip for single base-pair discrimination between the sequence of the wild type Kras gene and three of its common codon 12 mutants, which are frequently observed in the adenocarcinoma diseases. Here we use gold nanoparticles (AuNPs) in order to perform the single nucleotide polymorphism (SNP) assay without the need of temperature stringency. In the NBA chip, an array of 20-mer oligonucleotide probes was first created on the glass surface, and then a trace amount (2.5 fmol) of target oligonucleotides (20-mer and 60-mer) were introduced through the PDMS microchannels. DNA hybridization between the targets and their complementary probes was achieved at the intersections between microchannels and probe lines. The target oligonucleotides were previously loaded on the AuNP surfaces which established a competition between the comlementary probes and the AuNP surface for binding to the targets. This competition, in turn, enhances the hybridization stringency which disfavor sthe mismatch binding thus facilitates the SNP detection abilities. Using this technique fast and high-throughput multiple discrimination of Kras gene codon 12 was achieved at room-temperature using the NBA chip and the specificity of the method was proved to be as high as the temperature stringency method. 

Polyester Cloth–Based Hybridization Array System for Identification of Enterohemorrhagic Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157

A cloth-based hybridization array system (CHAS) was developed for the identification of foodborne colony isolates of seven priority enterohemorrhagic Escherichia coli (EHEC-7) serogroups targeted by U. S. food inspection programs. Gene sequences associated with intimin; Shiga-like toxins 1 and 2; and the antigenic markers O26, O45, O103, O111, O121, O145, and O157 were amplified in a multiplex PCR incorporating a digoxigenin label, and detected by hybridization of the PCR products with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. The EHEC-7 CHAS exhibited 100 % inclusivity and 100 % exclusivity characteristics with respect to detection of the various markers among 89 different E. coli strains, with various marker gene profiles and 15 different strains of non-E. coli bacteria.

1145 - Sensitive Detection of BRAF Mutations Using Mutant-Enriched PCR and Reverse-Hybridization Teststrips

ABSTRACT Background BRAF plays a key role in growth factor receptor induced signalling pathways. Somatic BRAF mutations are involved in oncogenesis and are found in various types of tumors, including colorectal, thyroid and skin cancer. Activating BRAF mutations confer resistance to anti-EGFR monoclonal antibody therapy. Moreover mutated BRAF is a prominent target of the drug vemurafenib. Materials and methods We have developed a reverse-hybridization StripAssay detecting nine BRAF mutations: c.1799T > C (V600A), c.1799_1800TG > AT (V600D), c.1799T > A (V600E), c.1799_1800TG > AA (V600E), c.1799T > G (V600G), c.1798_1799GT > AA (V600K), c.1798G > A (V600M), c.1798_1799GT > AG (V600R) and c.1801A > G (K601E). The test is based on mutant-enrichted PCR in the presence of a BRAF wild-type suppressor, followed by hybridization of PCR products to teststrips presenting a parallel array of allele-specific oligonucleotide probes. Bound sequences are visualized using streptavidin-alkaline phosphatase conjungate and colour substrates. The hybridization and detection steps can be carried out fully automated using commercially available instrumentation. Results Plasmid clones served as reference DNA templates to control for specificity. StripAssay performance was evaluated on genomic DNA obtained from cultured cell lines and formalin-fixed paraffine-embedded (FFPE) tissue. Using normal DNA spiked with serial dilutions of DNA from the BRAF-mutant tumor cell lines HT29 or IGR-1, the mutations c.1799T > A (V600E) and c.1798_1799GT > AA (V600K) were shown to be detectable at a level of 1%. Conclusions The simultaneous detection of nine different mutations with high sensitivity will make the StripAssay a very useful tool for the assessment of the BRAF mutation status of cancer patients. Disclosure All authors have declared no conflicts of interest.

Alterations in genes regulating cell cycle and apoptosis in high-grade urothelial carcinoma.

237 Background: Dysregulations of cell cycle and inhibition of apoptosis are crucial factors in tumorigenesis of multiple malignancies, including urothelial carcinoma (UC). Deletions of TP53 and CDKN2A and amplification of MDM2 have been observed in UC, but their exact frequency and functional consequence is less known. We sought to determine the frequency of copy number alteration (CNA) and mutations of genes that regulate cell cycle or apoptosis in a panel of 96 cases of high-grade UC (HGUC) of bladder. Methods: 96 frozen cases of HGUC were studied, including 11 bladder small cell carcinomas. DNA was isolated and analyzed for CNA by comparative genomic hybridization (CGH) using a one million oligonucleotide probe array from Agilent. The targeted genes included TP53, MDM2, CCND1, CCNE1, CDKN2A/B, E2F3 and Rb1. Traditional Sanger sequencing for mutations within TP53, Rb1, and CDKN2A was also performed. Results: The frequency of CNA and mutations are listed in the table. Overall, 54 of 96 cases (56%) showed CNA (45) or mutation (13). Deletion of CDKN2A/B and amplification of E2F3 were the most common alterations in cell cycle regulatory genes (13 cases each, 14%), followed by amplification of CCND1 (11 cases, 11%). There was no co-amplification of CCND1 and CCNE1 in any sample. Rb1 deletion was present in five cases. CNA in E2F3 and Rb1 were mutually exclusive in 14 of 16 cases (88%) and were both present in two cases only. Mutations in TP53 were noted in 13 cases and deletions in nine. Amplification of MDM2 was noted in four cases, none of which overlapped with TP53 deletions or mutations. Overexpression of E2F3 was significantly more common in small cell carcinoma (5/11) compared to conventional UC (8/85, p = 0.006). Conclusions: Regulators of cell cycle and apoptosis are amplified, deleted or mutated in more than half of cases (56%) of high-grade urothelial carcinoma. The overwhelming majority of these abnormalities are nonoverlapping. Amplification of E2F3 seems to be overrepresented in small cell carcinoma of bladder. [Table: see text] No significant financial relationships to disclose.

A simple PCR-based macroarray system for detection of multiple gene markers in the identification of priority enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) strains bearing the O antigenic determinants O157, O26, O111, O103, and O145 have a high rate of association with foodborne illness worldwide. To expand Canadian food inspection capability, a cloth-based hybridization array system (CHAS) was developed for the identification and characterization of priority EHEC. This method targets key virulence genes (eae, hlyA, vt1, and vt2) plus the rfbE gene specifying the O157 antigenic determinant, and the wzx genes specifying the O26, O111, O103, and O145 determinants. Multiplex PCR products incorporating a digoxigenin label were detected by hybridization with an array of specific oligonucleotide probes immobilized on a polyester cloth support, with subsequent immunoenzymatic assay of the captured amplicons. This method identified the relevant markers in 85 different strains bearing various combinations of the target genes (virulence and priority O-antigen markers). None of the target genes was detected in 26 different strains of other E. coli and non-E. coli bacteria. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the various markers among different bacterial strains. The CHAS demonstrated 100% inclusivity and 100% exclusivity characteristics, with respect to detection of the markers among various target and nontarget bacteria. The entire procedure could be completed in less than 5 h, and is useful for the identification of priority EHEC colonies isolated from foods by using enrichment culture techniques.

Sequencing-Based HLA Typing Using RNA

Genetic Engineering & Biotechnology NewsVol. 31, No. 5 TutorialSequencing-Based HLA Typing Using RNAAdvances in Extraction and Enzymology Open Door for RNA as Alternative to Genomic DNAMarrean Paul Theseira, DunnMarrean Paul Theseira, DunnMarrean Theseira is medical laboratory scientist and Paul Dunn (E-mail Address: paul.dunn@nzblood. co.nz)is clinical scientist at the Tissue Typing Laboratory at the New Zealand Blood Service. Web: www.nzblood.co.nz.Search for more papers by this authorPublished Online:1 Sep 2011https://doi.org/10.1089/gen.31.5.23AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byNovel Approaches and Technologies in Molecular HLA TypingHuman Leukocyte Antigen Typing Using a Knowledge Base Coupled with a High-Throughput Oligonucleotide Probe Array Analysis27 November 2014 | Frontiers in Immunology, Vol. 5 Volume 31Issue 5Mar 2011 Information© 2011 by GEN PublishingTo cite this article:Marrean Paul Theseira, Dunn.Sequencing-Based HLA Typing Using RNA.Genetic Engineering & Biotechnology News.Mar 2011.66-69.http://doi.org/10.1089/gen.31.5.23Published in Volume: 31 Issue 5: September 1, 2011PDF download

Design, Validation and Annotation of Transcriptome-Wide Oligonucleotide Probes for the Oligochaete Annelid Eisenia fetida

High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs). Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59%) probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1) does not require a major genomics core facility; (2) allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3) is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4) if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is particularly applicable to organisms with a wealth of EST sequences but unfinished genome.

Progressive but Previously Untreated CLL Patients with Greater Array CGH Complexity Exhibit A Less Durable Response to Chemoimmunotherapy

AbstractAbstract 2406The outcome for a given CLL patient is difficult to predict. While there are promising models, they require collation of multiple clinical and laboratory parameters, and it remains to be seen whether they will apply to typical CLL patients in the community. To further dissect out explanations for this dramatic clinical heterogeneity, we sought to understand genomic complexity of clonal B-cells as a possible explanation of clinical variability with specific application to genomic complexity as a predictor of therapeutic response and clinical outcome in CLL. Thus we wished to identified global gains and losses of genetic material in order to define copy-number abnormalities (CNA) in 48 clinically progressive CLL patients who were about to be treated on a chemoimmunotherapy protocol. This protocol was previously reported by us (Blood. 109:2007) and had an induction phase with pentostatin (2 mg/m2), cyclophosphamide (600 mg/m2) and rituximab (375 mg/m2) given every 3 weeks for 6 cycles and then responding patients were followed ever three months until relapse. In order to estimate CNA, we employed array-based comparative genomic hybridization (aCGH) using a one-million oligonucleotide probe array format on the leukemic B-cells from the 48 patients entering this trial. In those same patients, the aCGH data were compared to a) FISH detecxtable data using a panel for the common recurring genetic defects seen in CLL and b) to their clinical outcome on this trial. With aCGH we found that 288 CNA were identified (median of 4 per patient; range 0–32) of which 215 were deletions and 73 were gains. The aCGH method identified most of the FISH detected abnormalities with a complete concordance for 17p13.1- deletion (17p-) between aCGH and FISH. We also identified chromosomal gain or loss in ≥6% of the patients on chromosomes 3, 8, 9, 10, 11, 12, 13, 14 and 17. We found that CLL patients with ≥15 CNA had a significantly worse progression free survival (PFS) than patients with <15 CNA (p=0.004)(figure). Patients with ≥15 CNA also had a shorter duration of response than those with <15 CNA (p=0.0726). Of interest, more complex genomic features were found both in patients with a 17p13.1 deletion and in more favorable genetic subtypes such as 13q14.1. Thus, for 5 patients with >15 CNAs the following FISH patterns were seen: +12/13q14.1-x1/13q14.1 -x2, 13q14.1 ×1 (n=2), and 17p13.1 (n=2). In addition, a 17p- by FISH was positively associated with the number of CNA and total deletion size. The odds of having an overall response decreased by 28% (95% CI: 5–55%; p=0.015) with each additional CNA for the 17p13.1- patients. In addition to defining genomic complexity as the total number of CNA for each patient, we also defined complexity as the sum of the lengths of all interstitial chromosomal gains and losses. When defined as the total size of chromosomal gains or losses, genomic complexity was significantly associated with 17p13.1 and worse overall clinical response. In summary, this analysis utilized the global assessment of copy number abnormalities using a high-resolution aCGH platform for clinically progressive CLL patients prior to initiation of their treatment. One outcome was that we found higher genomic complexity was associated with shorter progression-free survival, reduced duration of response and predicted a poor response to treatment. In addition since we did find genomic complexity in more traditionally favorable FISH categories, such as 13q14.1 type defects, this may explain why some of the latter patients do not fare as well as might be expected even with aggressive chemoimmunotherapy approaches. This study adds information on the association between inferior trial response and increasing genetic complexity as CLL progresses. [Display omitted] Disclosures:Off Label Use: Pentostatin. Kipps: GlaxoSmithKline: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Genentech: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding; Genzyme: Research Funding; Memgen: Research Funding; Igenica: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi Aventis: Research Funding; Abbott Laboratories: Research Funding.

The UofL Microarray Facility: merger and new location

Background The UofL Microarray Facility is centered around the currently most advanced and comprehensive instrumentation for oligonucleotide array platforms by Affymetrix, Inc. and Agilent, Inc., both merged in the new location in CTR 227H-G. These technologies allow determination of global RNA gene expression and SNP (single nucleotide polymorphism) profiles based on hybridization of entire representative mRNA or genomic DNA populations to high-density arrays of gene-specific or mutation-specific oligonucleotide probes. Such global profiles can be obtained for tens of thousands of genes or hundreds of thousands of SNP loci from cell or tissue samples within one time-saving and standardized experiment. Comparative analysis of global gene expression or SNP patterns on biological material will provide crucial information on gene products and/or mutations which are involved in cellular functions, disease mechanisms, and therapy responses. Both cell culture, tissue, or microdissected cell material from human or various animal sources can be used for analyses. The objective of the UofL Microarray Facility is to ensure that investigators can efficiently adopt these state-of-the-art technologies for their basic science, clinical, and translational investigative goals. The Facility is registered as Academic Core Account and receives discounts for arrays and software licenses.

Cloth-based hybridization array system for the identification of Escherichia coli O157:H7

A simple cloth-based hybridization array system (CHAS) utilizing polyester cloth as the solid phase for DNA hybridizations has been adapted as a tool for the identification of presumptive verotoxigenic Escherichia coli O157:H7 colonies isolated from foods by standard culture techniques. Key indicator genes for this organism, rfbO157, fliCH7, vt1 and vt2 (encoding the O and flagellar antigenic determinants and verotoxin genes, respectively) were amplified in a multiplex PCR incorporating digoxigenin-dUTP, followed by hybridization of the amplicons with an array of specific oligonucleotide probes immobilized on polyester cloth, and subsequent immunoenzymatic assay of the bound digoxigenin label. This system includes a simple internal amplification control (IAC) to gauge PCR inhibition based on the incorporation of a primer pair with complementary 3′ ends, resulting in the generation of a unique “primer-dimer” detectable by hybridization with a specific capture probe immobilized on polyester cloth. The CHAS provided sensitive and specific detection of the E. coli O157:H7gene markers, exhibiting the expected patterns of reactivity with a panel of various target and non-target organisms.

Sensitive Detection of KRAS Mutations in Archived Formalin-Fixed Paraffin-Embedded Tissue Using Mutant-Enriched PCR and Reverse-Hybridization

Recently, evidence has emerged indicating that assessment of KRAS mutations before anti-epidermal growth factor receptor therapy improves outcome in patients with metastatic colorectal cancer (CRC). We report here a novel reverse-hybridization (RH) assay to screen for KRAS mutations in formalin-fixed paraffin-embedded colorectal tissue samples. We combined mutant-enriched PCR based on peptide nucleic acid clamping and RH of amplification products to nitrocellulose test strips that contained a parallel array of oligonucleotide probes targeting 10 frequent mutations in codons 12 and 13 of the KRAS gene. DNA mixing experiments, which included eight different tumor cell lines with known KRAS mutations, were performed to examine the sensitivity of mutation detection. All KRAS mutations present in tumor cell lines were unambiguously identified by the RH assay with 1% of each cell line DNA diluted in normal DNA. RH was then used to screen for KRAS mutations in 74 colorectal tumor and 4 normal control samples. Twenty-six (35%) of the 74 tumor samples showed KRAS mutations. No mutation was found in the four samples of normal colorectal tissue. DNA sequencing without previous mutant enrichment, however, failed to detect four (15%) out of 26 KRAS-positive formalin-fixed paraffin-embedded samples (FFPE). This finding suggests that even after microdissection, mutant sequences in a given DNA isolate can be rare and more sensitive methods are needed for mutation analysis.

ABO genotyping by duplex amplification and oligonucleotide arrays assay

Objective Research on the application feasibility of ABO genotyping for forensic identification by oligonucleotide arrays assay. Methods Oligonucleotide microarrays which detect three different SNPs in exon 6 and exon 7 for ABO genotyping were used. After hybridization wash, the arrays were scanned and fluorescence intensities were analyzed using microarray population studies on ABO was carried out in a sample of 115 unrelated Chinese Han individuals oligonucleotide arrays for genotype detection. The method was also applied to cases. Results Technique could identify six genotypes of ABO system and the results of GeneChip analyses confirmed by PCR–RFLP. According to the results of population studies, no significant deviations Hardy–Weinberg equilibrium could be found. The observed heterozygosity (H-obs) was 0.591. Expected heterozygosity (H-exp) was 0.616. The polymorphic information content (PIC) was the average exclusion probability in paternity testing for duos (PE (1)) was 0.188. The average exclusion probability in paternity testing for trios (PE(2)) was 0.344. The discrimination power 0.777. Conclusion The data and case application demonstrated that ABO typing by oligonucleotide probe arrays was a useful technique for paternity testing and individual identification.

Oligonucleotide Probe Array for p53 Gene Alteration Analysis in DNA from Formalin‐Fixed Paraffin‐Embedded Breast Cancer Tissues

Mutations in the TP53 tumor-suppressor (p53) gene represent the most common molecular changes in various malignancies, including breast cancer (BC). We sequenced the p53 gene in DNA extracted from archival paraffin-embedded BC tissues and compared the results obtained from direct sequencing with those obtained by oligonucleotide probe array (OPA). DNA was extracted from 34 samples. OPA correctly detected 13 genetic alterations in 14 cases, with a mutation frequency of 41.2%. No changes were detected in exons 3, 4, 9, 10, and 11 and no polymorphisms were found. Direct manual sequencing in which DNA was amplified by PCR showed 21 genetic mutations in 19 (55.9%) cases. Eight mutations were identified by both OPA and PCR methods. Although OPA detected fewer gene alterations than direct sequencing, the difference was not significant (P= 0.11). In conclusion, the OPA may be safely used to identify individual genetic variations of human p53 gene in BC specimens.

Sensor Array: Impedimetric Label‐Free Sensing of DNA Hybridization in Real Time for Rapid, PCR‐Based Detection of Microorganisms

AbstractA novel, impedance‐based electronic sensor format was used for label‐free, real‐time detection of microbial DNA on oligonucleotide probe arrays. Detection limits of 5–10 nM were achieved for single‐stranded, PCR‐amplified DNA targets. Hybridization selectivity yielded 9‐ to 12‐fold signal increases for specific targets, and the sensor arrays were re‐used multiple times without significant signal degradation. These and other features of the SHARP Laboratories of America (SLA) sensor array, such as its ability to acquire continuous measurements of DNA as it accumulates on the array surface, make it an attractive biosensor platform for field detection and monitoring of sentinel and/or pathogenic microorganisms.

Identification of unique and common low abundance tumour-specific transcripts by suppression subtractive hybridization and oligonucleotide probe array analysis

Most human cancers are characterized by genetic aberrations accompanied by altered expression and function of numerous genes. Applying genome-wide, microarray gene expression analysis to identify deregulated genes in different tumour types can provide potential gene candidates as diagnostic and prognostic tools and promising targets for drug development. However, the detection of deregulated genes with low levels of expression remains a major challenge. In this study, we have designed a strategy, termed modified suppression subtractive hybridization (mSSH), to identify genes encoding rare transcripts. The strategy entails incorporating the T(7)-promoter sequence at the 5' end of the noncoding cDNA strand during first strand cDNA synthesis to generate unidirectional antisense RNA from the resultant cDNA following conventional SSH. These transcripts are subsequently analysed by Affymetrix oligonucleotide gene arrays. Here, we have used five hepatocellular carcinoma (HCC), five breast carcinoma and four nasopharyngeal carcinoma (NPC) biopsies separately as testers and their corresponding normal biopsies as drivers to enrich for low abundance tumour type-specific transcripts. The total detectable number of probe sets following mSSH was reduced almost 10-fold in comparison to those detected for the same resected tumour tissues without undergoing subtraction, thus yielding a subtraction efficacy of over 90%. Using this experimental approach, we have identified 48 HCC-specific, 45 breast carcinoma-specific, and 83 NPC-specific genes. In addition, 115 genes were upregulated in all the three cancer types. When compared to gene-profiling data obtained without mSSH, the majority of these identified transcripts were of low abundance in the original cancer tissues. mSSH can therefore serve as a comprehensive molecular strategy for pursuing functional genomic studies of human cancers.

Rapid Authentication of Bupleurum Species using an Array of Immobilized Sequence-Specific Oligonucleotide Probes

Standardization and modernization of Chinese medicinal herbs are limited partially by misidentification of processed materials. Our goal was to develop an efficient method for verification of Chinese medicinal herbs, based on the variable sites of the rDNA internal transcribed spacer (ITS) region. We analyzed sequence differences in ITS of three Bupleurum species, B. kaoi Liu Chao et Chuang, B. falcatum L. and B. chinense DC., and developed a rapid detection method using a sequence-specific oligonucleotide probe (SSOP) array. The SSOP array, composed of poly-T tailed sequence-specific oligonucleotides, was hybridized to the digoxigenin (DIG)-labeled target ITS DNA of the Bupleurum species. The detected signals corresponded precisely to the specific sequences. This array provides a reliable and economical method for authenticating a large number of Chinese medicinal herbs. The short duration of the procedure (within 30 h) makes it an especially useful tool in verifying processed plant material.

Identification and Validation of a Novel Gene Signature Associated with the Recurrence of Human Hepatocellular Carcinoma

To improve the clinical management of human hepatocellular carcinoma (HCC) by accurate identification, at diagnosis, of patients at risk of recurrence after primary treatment for HCC. Two clinicopathologic variables available at diagnosis, vascular invasion and cirrhosis, together with molecular profiling using Affymetrix human HG-U133A and HG-U133B oligonucleotide probe arrays, were used to identify recurrent HCC disease. HCC patients presented clinically at diagnosis with vascular invasion and cirrhosis showed a high rate (78-83%) of developing recurrent disease within 6 to 35 months. In comparison, most of the HCC patients (80-100%) without vascular invasion and cirrhosis remained disease-free. However, the risk of recurrent disease for HCC patients with either vascular invasion or cirrhosis could not be accurately ascertained. Using a pool of 23 HCC patients with either vascular invasion or cirrhosis as training set, a 57-gene signature was derived and could predict recurrent disease at diagnosis, with 84% (sensitivity 86%, specificity 82%) accuracy, for a totally independent test set of 25 HCC patients with either vascular invasion or cirrhosis. On further analysis, the disease-free rate was significantly different between patients that were predicted to recur or not to recur in the test group (P = 0.002). We have presented data to show that by incorporating the status of vascular invasion and cirrhosis available at diagnosis for patients with HCC after partial curative hepatectomy and a novel 57-member gene signature, we could accurately stratify HCC patients with different risks of recurrence.