Universal Array Research Articles

Olive variety identification by ligation detection reaction in a universal array format

Molecular methodologies are increasingly being developed to assess the origin and authenticity of raw organic materials and processed food products. Here we describe the application of a microarray-based assay for single nucleotide polymorphisms (SNPs) identification in olive cultivars. The assay distinguishes alleles in a ligation detection reaction (LDR), with subsequent fluorescent detection by hybridization on a universal array (UA). The LDR-UA approach was used to detect 17 SNPs in olive genomic sequences previously amplified by PCR from fresh olive leaves. Genotype calls obtained with the LDR-UA were in full agreement with those determined by direct sequencing. The panel of 17 SNPs is sufficient to discriminate 49 olive varieties selected among the most widely cultivated for olive oil production in the Mediterranean area.

Development of a Single Nucleotide Polymorphism Genotyping Microarray Platform for the Identification of Bovine Milk Protein Genetic Polymorphisms

The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of αS1-casein, β-casein, κ-casein, and β-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the κ-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr136 and Ala148. This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.

Determination of CYP2D6, CYP2C9 and CYP2C19 genotypes with Tag–It™ mutation detection assays

Cytochrome P450 (CYP) genotyping can be used to prospectively identify individuals at risk for adverse drug reactions or therapeutic failure due to altered drug metabolism. Based on the specific CYP(s) affected, individuals may require less or more of a particular drug than people with unaffected CYP-mediated metabolism, or may be best managed by avoiding certain drugs entirely. Here we evaluated the Tag-It™ CYP mutation detection reagents (Tm Bioscience Corp.). As these reagents, based on a universal bead array, detect more than 20 clinically significant variants common to different ancestries, it was important to consider DNA from genetically diverse populations. Thus, we also report CYP2D6, CYP2C9 and CYP2C19 genotypes for DNA available through the Coriell Institute for Medical Research (NJ, USA). These samples represent individuals from Caucasian, Japanese, Chinese, Southeast Asian, African–American and Middle Eastern ancestry, and provide an excellent resource for evaluating and validating CYP genotyping methods. Using these samples, the Tag-It mutation detections assays reliably provided genotypes for CYP2D6, CYP2C9 and CYP2C19. The CYP2C9 and CYP2C19 assays were particularly robust and were easily implemented in our clinical laboratory. The CYP2D6 assay was somewhat less robust and could be improved by associating the 2850C>T variant with a specific allele, as well as by discriminating the allele affected when gene duplication is detected.

Exact and Approximation Algorithms for DNA Tag Set Design

In this paper, we propose new solution methods for designing tag sets for use in universal DNA arrays. First, we give integer linear programming formulations for two previous formalizations of the tag set design problem. We show that these formulations can be solved to optimality for problem instances of moderate size by using general purpose optimization packages and also give more scalable algorithms based on an approximation scheme for packing linear programs. Second, we note the benefits of periodic tags and establish an interesting connection between the tag design problem and the problem of packing the maximum number of vertex-disjoint directed cycles in a given graph. We show that combining a simple greedy cycle packing algorithm with a previously proposed alphabetic tree search strategy yields an increase of over 40% in the number of tags compared to previous methods.

A Structured Chitosan-Based Platform for Biomolecule Attachment to Solid Surfaces: Application to DNA Microarray Preparation

A structured chemical platform based on chitosan, an amine-rich polysaccharide, is presented as an alternative chemistry to functionalize solid support (in this case, glass slides) for grafting biomolecules. This approach has been adopted for generating arrays using amino-modified oligonucleotides with two different lengths (25-mer and 70-mer) for different purposes. Results using these chitosan-activated surfaces indicate high oligonucleotide loading capacity, good availability to hybridization against targets, and effectiveness in enzyme-mediated single nucleotide polymorphism (SNP) detection procedures by DNA polymerase and DNA ligase enzymes with low background. Universal arrays have been prepared and extensively used with excellent results in different applications. The chitosan-treated surfaces were also evaluated for their performance in a gene expression experiment.

Polymerase chain reaction/ligase detection reaction/hybridization assays using flow-through microfluidic devices for the detection of low-abundant DNA point mutations

We have microfabricated a flow-through biochip for the analysis of single base mutations in genomic DNA using two different materials: (1) a polycarbonate (PC) chip for performing a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using a universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for sequential PCR/LDR in a continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of gene fragments from genomic DNA; (2) mixing of the resultant PCR product with a LDR mixture via a Y-shaped passive micromixer and (3) ligation of two primers only when the particular mutation was present in the genomic DNA. A PMMA chip was employed as the microarray device, where zip code sequences (24-mer), which were complementary to sequences present on the discriminating primer, were micro-printed into fluidic channels embossed into the PMMA substrate. We successfully demonstrate the ability to detect one mutant DNA in 80 normal sequences with the integrated microfluidic device. The PCR/LDR/hybridization assay using the microchips performed the entire assay at a relatively fast processing speed: 18.7 min for PCR, 8.1 min for LDR, 5 min for hybridization, 10 min for washing and 2.6 min for fluorescence scanning (total processing time=ca. 50 min) with an order of magnitude reduction in reagents compared to bench-top formats.

Replication of DNA Microarrays from Zip Code Masters

This report describes a mechanical method for efficient and accurate replication of DNA microarrays from a zip code master. The zip code master is a DNA array that defines the location of oligonucleotides consisting of two parts: a code sequence, which is complementary to one or more of the zip codes, and the functional sequence, which is terminated with biotin. Following hybridization of the zip code to the code sequence, a replica surface functionalized with streptavidin is brought into conformal contact with the surface of the master. When the two surfaces are separated, the functional and code sequences are transferred to the replica, and the zip code remains on the surface of the master. Using this approach it is possible to prepare replica arrays having any configuration from a single, universal master array. Here we demonstrate that this approach can be used to replicate master arrays having up to three different sequences, that feature sizes as small as 100 microm can be replicated, and that master arrays can be used to prepare multiple replicas.

High-throughput DNA methylation profiling using universal bead arrays.

We have developed a high-throughput method for analyzing the methylation status of hundreds of preselected genes simultaneously and have applied it to the discovery of methylation signatures that distinguish normal from cancer tissue samples. Through an adaptation of the GoldenGate genotyping assay implemented on a BeadArray platform, the methylation state of 1536 specific CpG sites in 371 genes (one to nine CpG sites per gene) was measured in a single reaction by multiplexed genotyping of 200 ng of bisulfite-treated genomic DNA. The assay was used to obtain a quantitative measure of the methylation level at each CpG site. After validating the assay in cell lines and normal tissues, we analyzed a panel of lung cancer biopsy samples (N = 22) and identified a panel of methylation markers that distinguished lung adenocarcinomas from normal lung tissues with high specificity. These markers were validated in a second sample set (N = 24). These results demonstrate the effectiveness of the method for reliably profiling many CpG sites in parallel for the discovery of informative methylation markers. The technology should prove useful for DNA methylation analyses in large populations, with potential application to the classification and diagnosis of a broad range of cancers and other diseases.

Multiplexed SNP genotyping using nanobarcode particle technology

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.

Fabrication of DNA microarrays onto polymer substrates using UV modification protocols with integration into microfluidic platforms for the sensing of low-abundant DNA point mutations

We describe the microfabrication and operational characteristics of a simple flow-through biochip sensor capable of detecting low abundant point mutations in K- ras oncogenes from genomic DNA, which carry high diagnostic value for colorectal cancers. The biochip consisted of an allele-specific ligase detection reaction (LDR) coupled to a universal array for interrogating multiple mutations simultaneously from a clinical sample. The integrated sensing platform was micro-manufactured from two different polymers, polycarbonate, PC, which was used for the LDRs, and poly(methyl methacrylate), PMMA, which was used to build the microarray. Passive elements were hot embossed into the PC and PMMA microchips and then, the chips assembled into a three-dimensional architecture with the interconnect fabricated from an elastomer, poly(dimethylsiloxane), PDMS, to produce a leak-free connection between the biochips. The array in PMMA was produced using a photomodification process, which involved three steps; (1) UV (254 nm) exposure of the polymer surface; (2) EDC coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond) and; (3) washing of the surface. The LDR/hybridization flow-through biochip performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time = 19.1 min) and could screen multiple mutations simultaneously for high throughput applications at a level of one mutant sequence in 100 wild-type sequences.

Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods

A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in genetically modified soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the efficiency of all reactions. This multiplex PCR has constituted the basis for an efficient platform for genetically modified organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High specificity and sensitivity were obtained.

Gene expression profiling in formalin-fixed, paraffin-embedded (FFPE) benign and cancerous prostate tissues using universal bead arrays

9526 Background: Archival tissues from patients with known clinical history have become an invaluable resource for gene expression analysis for tumor classification and clinical outcome prediction. These samples may be studied using qPCR, but RNA extracted from FFPE tissues is often significantly degraded, making them difficult to use in microarray analyses. Methods: We have developed the DASL assay, a flexible, sensitive and reproducible expression profiling method for parallel analysis of hundreds of genes in FFPE samples using universal bead arrays. The method can tolerate partial RNA degradation because it does not depend on an intact poly-A tail for cDNA synthesis. Tissue- and cancer-specific expression profiles have been obtained with as little as 50 ng total RNA derived from FFPE tissues that had been stored up to 11 years (Bibikova, et al., Am. J. Pathol. 165:1790–1807, 2004). Results: We performed a profiling study of 174 benign or cancerous prostate FFPE tissues retrieved from the VA San Diego Healthcare System. A total of 512 prostate cancer-related genes selected from the literature were monitored at 3 probes per gene. Despite RNA degradation, highly reproducible expression profiles were obtained for these samples (average r2 = 0.98). Expression data were used to identify tumor- and stroma-specific genes, which were then included in a sample classification scheme. Resulting gene panels correlated with clinical parameters such as tumor type, tumor grade, therapeutic response, and patient survival. In additional studies using laser capture microdissection of FFPE tissues, increased expression of tumor- and stroma-specific genes were measured in the correct cell types, confirming the results based on heterogeneous tissues. Conclusions: Putative prostate cancer biomarkers were identified using the DASL expression assay and RNAs derived from FFPE patient samples. In combination with the high-throughput capability of the bead array-based platform, the DASL assay may be used to monitor hundreds of genes in hundreds of samples at once. These data suggest that FFPE tissue archives, with known patient outcomes, can now be accessed for microarray expression analysis. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Illumina Illumina

Ligase Detection Reaction/Hybridization Assays Using Three-Dimensional Microfluidic Networks for the Detection of Low-Abundant DNA Point Mutations

We have fabricated a flow-through biochip assembly that consisted of two different microchips: (1) a polycarbonate (PC) chip for performing an allele-specific ligation detection reaction (LDR) and (2) a poly(methyl methacrylate) (PMMA) chip for the detection of the LDR products using an universal array platform. The operation of the device was demonstrated by detecting low-abundant DNA mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. The PC microchip was used for the LDR in a continuous-flow format, in which two primers (discriminating primer that carried the complement base to the mutation being interrogated and a common primer) that flanked the point mutation and were ligated only when the particular mutation was present in the genomic DNA. The miniaturized reactor architecture allowed enhanced reaction speed due to its high surface-to-volume ratio and efficient thermal management capabilities. A PMMA chip was employed as the microarray device, where zip code sequences (24-mers), which were complementary to sequences present on the target, were microprinted into fluidic channels embossed into the PMMA substrate. Microfluidic addressing of the array reduced the hybridization time significantly through enhanced mass transport to the surface-tethered zip code probes. The two microchips were assembled as a single integrated unit with a novel interconnect concept to produce the flow-through microfluidic biochip. A microgasket, fabricated from an elastomer poly(dimethylsiloxane) with a total volume of the interconnecting assembly of <200 nL, was used as the interconnect between the two chips to produce the three-dimensional microfluidic network. We successfully demonstrated the ability to detect one mutant DNA in 100 normal sequences with the biochip assembly. The LDR/hybridization assay using the assembly performed the entire assay at a relatively fast processing speed: 6.5 min for on-chip LDR, 10 min for washing, and 2.6 min for fluorescence scanning (total processing time 19.1 min) and could screen multiple mutations simultaneously.

Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations

We have developed a simple ultraviolet (UV)-photomodification protocol using poly(methyl methacrylate) and polycarbonate to produce functional scaffolds consisting of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes to these surface groups through carbodiimide coupling. Use of the photomodification procedure coupled to microfluidics allowed for the rapid generation of medium-density DNA microarrays. The method reported herein involves the use of poly(dimethylsiloxane) microchannels reversibly sealed to photomodified poly(methyl methacrylate) surfaces to serve as stencils for patterning the oligonucleotide probes. After array construction, the poly(dimethylsiloxane) stencil is rotated 90° to allow interrogation of the array using microfluidics. The photomodification process for array fabrication involves only three steps: (1) broadband UV exposure of the polymer surface, (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond), and (3) washing of the surface. The density of probes attached to this activated surface was found to be ∼41 pmol cm −2, near the steric-saturation limit for short oligonucleotide probes. We demonstrate the use of this procedure for screening multiple KRAS2 mutations possessing high diagnostic value for colorectal cancers. A ligase detection reaction/universal array assay was carried out using parallel detection of two different low-abundant DNA point mutations in KRAS2 oncogenes with the allelic composition evaluated at one locus. Four zip code probes immobilized onto the poly(methyl methacrylate) surface directed allele-specific ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of cross-hybridization or misligation.

A Microarray Platform for Parallel Detection of Five Transgenic Events in Foods: A Combined Polymerase Chain Reaction−Ligation Detection Reaction−Universal Array Method

We recently developed a multiplex polymerase chain reaction (PCR) system for the simultaneous detection of four transgenic maize (MON810, Bt176, Bt11, and GA21), one transgenic soybean (Roundup Ready), and two control genes (lectin and zein). Because PCR can lead to ambiguous interpretations due to low specificity, we have developed the ligation detection reaction (LDR) combined with a universal array as a molecular tool to confirm results of PCR analysis. Here, we describe the PCR-LDR-universal array procedure and demonstrate its specificity in revealing the presence of transgenic DNA in experimental samples, raw materials, and commercial foodstuffs.

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms.

We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs). Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers. The products were then sorted on the Tag-It array and detected by use of the Luminex xMAP system. Genotypes were also determined by sequencing. Empirical validation of the universal array showed that the highest nonspecific signal was 3.7% of the specific signal. Patient genotypes showed 100% concordance with direct DNA sequencing data for 736 SNP determinations. The Tag-It microsphere-based universal array platform is a highly accurate, multiplexed, high-throughput SNP-detection platform.

Polymorphism analysis within the HLA-A locus by universal oligonucleotide array.

Human leukocyte antigen (HLA) class I genes present some of the most complex single nucleotide polymorphism (SNP) patterns in the human genome. HLA typing is therefore extremely challenging. In this article, we use the ligation detection reaction (LDR) combined with a universal array (UA) as a robust and efficient method to analyze SNPs within the HLA-A region that includes HLA-A alleles of interest for immunotherapy in tumor diseases. The LDR, combined with a UA platform, has been optimized for the detection of 27 alleles distributed within exons 2 and 3 of HLA-A. The assay involves the amplification by PCR of the HLA-A genomic region (1,900 bp), the cycled ligation reaction, followed by the capture of ligated products through hybridization onto a UA. Each slide was designed to allow the detection of up to eight samples in parallel. The PCR/LDR/UA HLA-A assay was evaluated by analyzing 62 individuals (31 homozygous and 31 heterozygous) previously typed by direct sequencing. We demonstrate that the microarray genotyping procedure described here is a robust and efficient method for unambiguous detection of HLA alleles. HLA genotyping by PCR/LDR/UA is in perfect agreement with typing obtained by direct sequencing. Our results clearly demonstrate that the combination of enzymatic processing (LDR) and a demultiplexing hybridization onto a UA is a robust tool for SNP discrimination within the highly polymorphic HLA region. We demonstrate the specificity and efficiency of such an approach, suggesting the feasibility of a PCR/LDR/UA low resolution HLA typing procedure.

Universal RNA reference materials for gene expression.

A workshop entitled "Metrology and Standards Needs for Gene Expression Technologies: Universal RNA Standards" was held in March 2003 to define the requirements for standardizing RNA-based molecular assays, specifically microarray and quantitative reverse-transcriptase-PCR technologies. NIST sponsored the workshop, and participants represented government, industry, academia, and clinic. Workshop participants concluded that as a first step, two RNA reference materials could be defined that would help in standardization of gene-expression technologies: an Assay Process Reference Material, and a Universal Array Hybridization Reference Material. The specific characteristics of these two standardized materials were broadly outlined. The Assay Process Material was proposed to be a pool of 96 expressed human sequences of defined composition, cloned in a defined vector and pooled in prescribed ways. The Universal Array Hybridization Material was defined as a pool of 12 "alien" synthetic sequences not expressed in any known genome to be used to control for variability in array hybridization methods. Work is underway at NIST and among members of the gene expression array community to further define these materials and make them available.

Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.

Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.