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Combinatorial multiplex assay format using electronic microchip arrays and its potential application in complex cancer diagnostics.

A key challenge in cancer treatment and prevention is early disease detection, thus facilitating effective therapeutic intervention to improve quality of life and survival. With molecular genetics playing an increasingly important role in solid tumor diagnostics, the identification of biomarkers that act as signatures of specific cancers at various developmental stages is critical to enable early definitive detection and diagnosis (1). Established cancer biomarkers can be quickly screened and detected by sensitive molecular genotyping to assess cancer risk and afford the earliest therapeutic intervention possible. Such biomarkers may also render meaningful prognostic information as well as assist in the selection of individual treatment regimens. As cancer biomarkers are discovered and characterized, the ability to quickly and economically screen large numbers of patients for a wide variety of markers is essential. This capability is further underscored by the complex and varied mutation profiles associated with cancer markers.

In the present study, a combination of well-characterized gene mutations encompassing single-nucleotide polymorphisms, microdeletions, and insertions commonly found in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was used to provide proof of principle for a method capable of simultaneously identifying multiple specific alleles by use of electronically controlled microchips. Mutation regions are amplified and applied to a single microchip test site addressed with multiple biotinylated oligonucleotide capture probes. Fluorescently labeled reporter probes specific to wild-type or mutant alleles are then hybridized to the complex to provide precise genotyping. This method does not identify, but only detects the presence of any mutant alleles in a specimen; however, it permits the simultaneous screening of a large cohort of patients for a large number of different mutations. This rapid high-throughput screening platform, designated combinatorial multiplexing, can be applied to any disease-associated genes or biomarkers, especially those with complex genotype profiles, to aid in clinical diagnosis.

We selected 11 well-characterized mutations encompassing seven regions in CFTR and designed primers for PCR amplification based on sequences obtained from GenBank (accession nos. M55106-M55131). The primer sequences are listed in Table 1. Mutations examined were G085E, 405+3A[right arrow]C, 711+1G[right arrow]T, R334W, R347H, R347P, A455E, [Delta]F508, 2184delA, 2307insA, and 3659de1C (2). Each amplicon was amplified from 15 ng of genomic DNA in a 50-[micro]L reaction consisting of 1 x PCR buffer (Invitrogen), 1.8 mM Mg[Cl.sub.2], 400 [micro]M deoxynucleotide triphosphates, 400 nM each primer, and 5 U of Platinum Pfx DNA polymerase (Invitrogen). Thermal cycling conditions were as follows: 95 [degrees]C for 3 min; 35 cycles of 95 [degrees]C for 30 s, 55 [degrees]C for 30 s, and 72 [degrees]C for 45 s; and final extension at 72 [degrees]C for 2 min. PCR products were desalted through Multiscreen-PCR filter plates (Millipore) and analyzed on 1.5% agarose gels.

Capture oligonucleotides specific for each selected mutation were synthesized with 3' biotinylated ends to permit permanent addressing onto Nanochip[TM] (Nanogen) arrays, which feature a protective agarose permeation layer containing streptavidin (3). Capture and reporter oligonucleotides were designed to permit amplicon-directed juxtaposition of the 3'-terminal base of the reporter to the 5'-terminal base of the capture when a positive hybridization match occurs. This method takes advantage of the premise that base stacking stabilizes DNA hybridization such that matched reporter binding is preferentially more stable than mismatched reporter binding (4). Only in the presence of base stacking, when capture and reporter oligonucleotides are in direct apposition and paired to the patient amplicon, will the hybrid be stabilized. Capture/ reporter allele sets were first individually assessed to confirm their ability to specifically detect and discriminate between mutant and wild-type specimens. Capture probes were diluted to a concentration of 0.5 [micro]mol/L in 50 mmol/L histidine and electronically addressed to selected sites on the microchip by use of mapping protocols created from instrument software (5). Sites were also biased with histidine alone to serve as background controls. Appropriate target amplicons were denatured at 95 [degrees]C for 5 min and quickly cooled on ice before hybridization. A 5-[micro]L sample of each denatured amplicon in 50 mmol/L histidine was then electronically hybridized to the relevant capture-containing sites on the chip.

After amplicon hybridization, chips were manually washed three times with high-salt buffer [50 mmol/L sodium phosphate (pH 7.4), 500 mmol/L NaCl]. Pairs of allele-specific reporters labeled with either Cy3 (wild type) or Cy5 (mutant) fluorophore were mixed equally at 0.5 [micro]mol/L to yield a combined 1 [micro]mol/L in high-salt buffer. Reporter solutions were applied to the chip and hybridized passively for 3 min at room temperature, followed immediately by three more washes in high-salt buffer. Thermal discrimination and fluorescence detection were then performed in the fluorescent reader instrument. Chips were loaded into the reader and washed with low-salt buffer (50 mmol/L sodium phosphate, pH 7.4); an initial fluorescent image was then taken by use of separate lasers for each fluorophore. The capture/target/reporter complex was then thermally denatured to achieve precise discrimination between matched and mismatched alleles, followed by washes in low-salt buffer at the increased temperature. A final fluorescent scan of the array was taken, and background controls were subtracted from each pad's signal for final quantification. Optimal discrimination was achieved in all of the alleles at a temperature range of 27-28 [degrees]C.

When analyzing individual alleles, we scored fluorescence hybridization patterns by the criteria that wild-type samples meet or exceed a 5:1 threshold ratio of Cy3 to Cy5 signal, that heterozygous samples exhibit a 3:1 ratio in either direction, that homozygous mutant samples have a 5:1 ratio of Cy5 to Cy3 signal, and that the signal-to-noise ratios for any reporter be at least 5:1. These genotypes were assigned using the manufacturer's recommended biallelic fluorescence intensity ratios, and no genotype designations were made for fluorescence intensity ratios between 1:3 and 1:5. Homozygous wild-type alleles were all robustly bound by Cy3-labeled reporter, whereas homozygous mutant alleles were bound only by Cy5-labeled reporter. Heterozygous amplicons exhibited both Cy3 and Cy5 signals at equivalent intensities, identifying the presence of both wild-type and mutant alleles. Control captures hybridized to nonspecific amplicons or reporters appropriately showed background fluorescence, demonstrating the high specificity of each individual capture/amplicon/reporter group. DNA samples with known cystic fibrosis mutations were previously genotyped and provided by ARUP Laboratories.

We next addressed arrays with diluted amounts of capture probes to simulate multiplexed assay conditions. Reduction of each capture's quantity volumetrically allows for more captures to be combined at once, while also lowering reagent cost. The increase in the number of different captures present is offset by the final molar quantity of each capture bound to the array surface. To assess assay sensitivity when captures are diluted out by competing captures, we addressed mixtures of two captures at molar ratios of 1:29 and 29:1 to simulate a potential 30-capture mixture. After hybridization of the captures' two corresponding wild-type amplicons, Cy3 fluorescence intensity for the more concentrated allele was robust, whereas signal intensity for the more dilute allele was slightly less but still unmistakably detectable. The same results were obtained when the molar ratios of the two captures were reversed and when different captures were used.

Multiplex assays were prepared by addressing nine different captures simultaneously onto single pads in which the concentration of each capture probe in the mixture was 83.3 nmol/L. A mixture of wild-type amplicons corresponding to all captures represented on the multiplexed sites was hybridized, and reporter sets were individually applied to assess each capture/amplicon/reporter hybrid independently. For each capture present in the multiplex, another multiplex pad in which that particular capture was absent from the mixture was prepared as a negative control for target specificity. For each multiplexed capture, the corresponding wild-type amplicon was detected and correctly genotyped, whereas the control multiplex pad (in which the capture in question was absent) exhibited fluorescence intensities near that of background. Systematic application of each reporter set in this manner confirmed the specificity of each capture/amplicon/reporter complex in a multiplex format. Use of heterozygous or mutant amplicons in the multiplex assay also led to their successful detection and accurate scoring as such, demonstrating that the multiplex format is capable of discriminating between genotypes. Fig. 1 shows the fluorescence patterns of various individual alleles in a heterozygous carrier for G085E in the multiplexed format.

Although this multiplex assay successfully combined 9 mutation alleles on a single test site, data from our experiments using diluted captures suggest that it is theoretically possible to screen for at least 30 mutations simultaneously without a considerable loss in assay sensitivity. Fluorescence intensity was reduced ~30% for a capture diluted 1:29 by a competing capture, but the signal-to-noise ratios remained >20:1 and fluorescence genotyping criteria were preserved. Because the objective of the multiplex screening assay is to simply detect the presence of any mutant alleles, the unambiguous strength of a single positive mutant signal is sufficient to identify the presence of the mutation. To better circumvent potential problems associated with nonspecific oligonucleotide cross-hybridization in a highly complex mixture, however, it may be more practical to limit the number of mutations screened on a test site to 15, considering that each microchip array offers 100 sites.

[FIGURE 1 OMITTED]

Conventional methods of mutation detection are often time-consuming and laborious, making detection of diseases with complex mutation profiles a monumental task. Our results demonstrate that complex multiallelic mutation detection can be rapidly and accurately achieved on a prefabricated microchip array, making it highly amenable for routine detection of the diverse assortments of deleterious alterations found in many cancer markers and heritable diseases. Collectively, our methodology imparts several potential advantages over existing mutation detection systems, including high throughput and rapid turnaround, which may in turn lower genotyping costs.

In summary, combinatorial multiplexing is an efficient and, more importantly, accurate method for multiple mutation detection that allows for the potential to simultaneously and rapidly screen a large number of patients for the presence of many different mutations.

References

(1.) Srinivas PR, Kramer BS, Srivastava S. Trends in biomarker research for cancer detection. Lancet Oncol 2001;2:698-704.

(2.) Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;2:149-54.

(3.) Gilles PN, Wu DJ, Foster CB, Dillon PJ, Chanock SJ. Single nucleotide polymorphic discrimination by an electronic dot blot assay on semiconductor microchips. Nat Biotechnol 1999;17:365-70.

(4.) Radtkey R, Feng L, Muralhidar M, Duhon M, Canter D, DiPierro D, et al. Rapid, high fidelity analysis of simple sequence repeats on an electronically active DNA microchip. Nucleic Acids Res 2000;28:E17.

(5.) Edman CF, Raymond DE, Wu DJ, Tu E, Sosnowski RG, Butler WF, et al. Electric field directed nucleic acid hybridization on microchips. Nucleic Acids Res 1997;25:4907-14.

Phillip S. Kim, * Denice K. Tai, and Kan V. Lu

KnowledGENE, Inc., Department of Research and Development, 13206 Estrella Ave., Suite C, Gardena, CA 90248;

* author for correspondence: fax 310-533-0506, e-mail pkim@knowledgene.com
Table 1. PCR amplicons and primer sequences.

PCR amplicon position CF mutations covered

Exon 3-Intron 3 G085E, 405+3A[right arrow]C

Exon 5-Intron 5 711+1G[right arrow]4T

Exon 7 R334W, R347H, R347P

Exon 9 A455E

Exon 10 [Delta]F508

Exon 13 2184delA, 2307insA

Exon 19 3659delC

PCR amplicon position Primer sequences (a)

Exon 3-Intron 3 F: 5'-CGATGTTTTTTCTGGAGATTTATGT-3'
 R: 5'-ATCCTTACTAGAGTTTTAGGTGGTT-3'
Exon 5-Intron 5 F: 5'-GACAACTTGTTAGTCTCCTTTCC-3'
 R: 5'-ACATGTACGATACAGAATATATGT-3'
Exon 7 F: 5'-TGCACTAATCAAAGGAATCA-3'
 R: 5'-TCCTAGTATTAGCTGGCAA-3'
Exon 9 F: 5'-TACTCCTGTCCTGAAAGATA-3'
 R: 5'-TACACCCATACATTCTCCTA-3'
Exon 10 F: 5'-ATGATTATGGGAGAACTGGA-3'
 R: 5'-TATAATTTGGGTAGTGTGAAGGGT-3'
Exon 13 F: 5'-AAAGAAGAAATTCAATCCTAAC-3'
 R: 5'-AGTGTGTCATCAGGTTCAGG-3'
Exon 19 F: 5'-ATGCGATCTGTGAGCCGA-3'
 R: 5'-ATTTCCACCTTCTGTGTATTTTGCT-3'

PCR amplicon position Amplicon size, bp

Exon 3-Intron 3 205

Exon 5-Intron 5 254

Exon 7 286

Exon 9 206

Exon 10 238

Exon 13 365

Exon 19 214

(a) F, forward; R, reverse.
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Title Annotation:Abstracts of Oak Ridge Posters
Author:Kim, Phillip S.; Tai, Denice K.; Lu, Kan V.
Publication:Clinical Chemistry
Date:Oct 1, 2002
Words:1993
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