High throughput assay system

#This invention relates e.g. to compositions, apparatus and methods useful for concurrently performing multiple biological or chemical assays, using repeated arrays of probes. A plurality of regions each contains an array of generic anchor molecules. The anchors are associated with bifunctional linker molecules, each containing a portion which is specific for at least one of the anchors and a portion which is a probe specific for a target of interest. The resulting array of probes is used to analyze the presence of one or more target molecules which interact specifically with the probes. The invention relates to diverse fields distinguished by the nature ofthe molecular interaction, including but not limited to pharmaceutical drug discovery, molecular biology, biochemistry, pharmacology and medical diagnostic technology.

#Pluralities of molecular probes arranged on surfaces or “chips” have been used in a variety of biological and chemical assays. Assays are performed to determine if target molecules of interest interact with any of the probes. After exposing the probes to target molecules under selected test conditions, detection devices determine whether a target molecule has interacted with a given probe.

#These systems are useful in a variety of screening procedures for obtaining information about either the probes or the target molecules. For example, they have been used to screen for peptides or potential drugs which bind to a receptor of interest, among others; to screen samples for the presence of, for example, genetic mutations, allelic variants in a population, or a particular pathogen or strain of pathogen, among many others; to study gene expression, for example to identify the mRNAs whose expression is correlated with a particular physiological condition, developmental stage, or disease state; etc.

#FIG. 1 illustrates a design scheme for oligonucleotides, in which a linker 1 contains a portion that is specific for anchor 1 and another portion (a probe) that is specific for target mRNA 1, and in which a labeled detection probe 1 is specific for a sequence of target MRNA 1 which is different from the sequence of the target-specific portion of the linker.

#FIG. 2 illustrates a surface which comprises 15 test regions, each of which comprises an array of six anchor oligonucleotides.

#FIG. 3 illustrates the design of a linker for a receptor binding assay, in which the anchor-specific portion of the linker is associated with the probe portion (the receptor protein) via biotin and streptavidin molecules, and in which a ligand specific for the receptor is labeled with a fluorescent labeling molecule. B: Biotin. SA: Streptavidin. Rec: Receptor protein. Ligand: a natural or synthetic ligand for the receptor. *: a fluorescent labeling molecule attached to the Ligand.

#Hybridization Specificity (see FIG. 10)

#A generic MAPS plate was produced by using an inkjet dispenser, the Pixus system (Cartesian Technologies, Inc., Irvine, Calif.) to form an identical grid of DNA within each well of a microtiter plate. All oligonucleotides were purchased from Biosource International (Camarillo, Calif.). For this plate, seven different oligonucleotide anchors were dispensed within each well in the pattern shown as the Key (left side of the figure). Each oligonucleotide was dispensed as a 10 nanoliter droplet to two spots, from a 2 uM solution containing 500 mM sodium phosphate pH 8.5 and 1 mM EDTA to the wells of a DNA Bind plate (Corning Costar), and allowed to dry. After attachment, wells were blocked with 50 mM Tris pH 8, and then oligonucleotide that had not covalently attached to the surface was washed away with 0.1% SDS in 5× SSP buffer. To the washed plate fluorescently labeled linker oligonucleotides were added and allowed to hybridize in 6× SSPE with 0.1% Triton X-100 at room temperature for thirty minutes. This is a preferred protocol for attachment of linkers. The linker oligonucleotides were cy5-derivatized during synthesis, and were complementary in 25 base-pair segments to specific anchoring oligonucleotides. The sequences of the seven anchors and linkers were as follows (all shown 3′ to 5′):

##1 Anchor*: SEQ ID:1 TCCACGTGAGGACCGGACGGCGTCC Linker** SEQ ID:2 GTCGTTTCCATCTTTGCAGTCATAGGATACTGAGTGGACGCCGTCCGGTCCTCACG TGGA RNA mimic(mouse C-jun): SEQ ID:3 CTATGACTGCAAAGATGGAAACGACGATACTGAGTTGGACCTAACATTCGATCTC ATTCA Detector Oligonucleotide*** SEQ ID:4 TGAATGAGATCGAATGTTAGGTCCA #2 Anchor*: SEQ ID:5 CACTACGGCTGAGCACGTGCGCTGC Linker** SEQ ID:6 CTAGGCTGAAGTGTGGCTGGAGTCTGCAGCGCACGTGCTCAGCCGTAGTG RNA mimic (mouse MIP-2): SEQ ID:7 AGACTCCAGCCACACTTCAGCCTAGGATACTGAGTCTGAACAAAGGCAAGGCTAA CTGAC Detector Oligonucleotide*** SEQ ID:8 GTCAGTTAGCCTTGCCTTTGTTCAG #3 Anchor*: SEQ ID:9 GTCAGTTAGCCTTGCCTTTGTTCAG Linker** SEQ ID:10 ACCATGTAGTTGAGGTCAATGAAGGGCGCTCCCACAACGCTCGACCGGCG RNA mimic (mouse GAPDH): SEQ ID:11 CCTTCATTGACCTCAACTACATGGTGATACTGAGTGGAGAAACCTGCCAAGTATG ATGAC Detector Oligonucleotide*** SEQ ID:12 GTCATCATACTTGGCAGGTTTCTCC #4 Anchor*: SEQ ID:13 GAACCGCTCGCGTGTTCTACAGCCA Linker** SEQ ID:14 CTACCGAGCAAACTGGAAATGAAATTGGCTGTAGAACACGCGAGCGGTTC RNA mimic (mouse L32 protein): SEQ ID:15 ATTTCATTTCCAGTTTGCTCGGTAGGATACTGAGTGAGTCACCAATCCCAACGCCA GGCT Detector Oligonucleotide*** SEQ ID:16 AGCCTGGCGTTGGGATTGGTGACTC #5 Anchor*: SEQ ID:17 CTCGTTCCGCGTCCGTGGCTGCCAG Linker** SEQ ID:18 CTGGCAGCCACGGACGCGGAACGAG #6 Anchor*: SEQ ID:19 CGGTCGGCATGGTACCACAGTCCGC Linker** SEQ ID:20 GCGGACTGTGGTACCATGCCGACCG #7 Anchor* SEQ ID:21 GCGCGCCGCGTTATGCATCTCTTCG Linker** SEQ ID:22 CGAAGAGATGCATAACGCGGCGCCG *Anchors were synthesized with C12 spacer with amide at the 5′ end **Linkers were synthesized with Cy5 attached at the 5′ end ***Detector Oligonucleotides were synthesized with biotin attached at the 5′ end