Invented by Francis Barany, George Barany, Robert P. Hammer, Maria Kempe, Herman Blok, Monib Zirvi, Cornell Research Foundation Inc

The market for detection of nucleic acid sequence variations using the ligase detector reaction with addressable arrays has been growing rapidly in recent years. This technology has revolutionized the way we detect genetic mutations and variations, allowing for faster and more accurate diagnosis of diseases and disorders. The ligase detector reaction (LDR) is a powerful tool for detecting single nucleotide polymorphisms (SNPs) and other sequence variations in DNA. It works by using a DNA ligase enzyme to join two DNA strands together, only if they match perfectly at a specific point. This allows for highly specific detection of sequence variations, even in complex mixtures of DNA. Addressable arrays are a key component of this technology, allowing for high-throughput analysis of large numbers of samples. These arrays consist of thousands of tiny spots, each containing a specific DNA sequence that can be used to detect variations in the target DNA. The market for LDR-based detection of nucleic acid sequence variations is driven by a number of factors, including the increasing prevalence of genetic diseases and disorders, the growing demand for personalized medicine, and the need for faster and more accurate diagnostic tools. One of the key applications of this technology is in the field of cancer diagnosis and treatment. By detecting specific mutations in cancer cells, doctors can tailor treatments to individual patients, improving outcomes and reducing side effects. Other applications include genetic testing for inherited diseases, infectious disease diagnosis, and forensic analysis. The ability to detect sequence variations with high accuracy and specificity has the potential to revolutionize many areas of medicine and science. The market for LDR-based detection of nucleic acid sequence variations is highly competitive, with a number of companies offering innovative products and services. Some of the key players in this market include Illumina, Thermo Fisher Scientific, Qiagen, and Roche. As the technology continues to evolve and improve, we can expect to see even greater demand for LDR-based detection of nucleic acid sequence variations. This will drive innovation and competition in the market, leading to new products and services that will benefit patients and researchers alike.

The Cornell Research Foundation Inc invention works as follows

The present invention describes a method of identifying one or several sequences that differ by one or multiple single base changes, inserts, deletions or translocations in a plurality target nucleotide sequencing. The method comprises a ligation, capture, and detection phases. The ligation phase uses a ligation detection process between one oligonucleotide prob, which has a target-specific section and an addressable array?specific portion, and another oligonucleotide pro, with a target-specific section and a detectable tag. The ligation phase is followed by the capture phase. This involves hybridizing the ligated Oligonucleotide Probes to a solid Support with an array immobilized capture Oligonucleotides, at least one of which is complementary to the addressable array specific portion. After the capture phase is completed, a detection phase is performed to detect the labels of ligated Oligonucleotide Probes that have been hybridized to the solid Support.

Background for Detection nucleic acid sequence variations using the ligase detector reaction with addressable arrays.

Detection Sequence Differences

For practical identification of individuals, large-scale multiplex analysis is required of highly polymorphic loci (Reynolds et al., Anal. Chem., 63:2-15 (1991)), for organ-transplant donor-recipient matching (Buyse et al., Tissue Antigens, 41:1-14 (1993) and Gyllensten et al., PCR Meth. Appl. 1:91-98 (1991), for pre-natal counseling and genetic disease diagnosis (Chamberlain et. al. Nucleic Acids Res. 16:11141-11156 (1988), and L. C. Tsui Human Mutat. 1:197-203 (1992), and the study oncogenic mutations (Hollstein et. al. Science, 253 :49-53 (1991). The cost-effectiveness for infectious disease diagnosis using nucleic acid analysis is directly related to panel testing’s multiplex scale. These applications rely on discrimination of single base differences at multiple, sometimes close-knit loci.

There are many DNA hybridization methods that can detect the presence of selected polynucleotide regions in samples with a lot of sequence regions. A simple method that relies on fragment capture is hybridization to immobilized probes. This allows for the capture of a fragment with a specific sequence. Hybridization to another probe that contains a detectable reporter moiety can label the fragment captured.

Southern blotting is another widely-used method. This method uses a mixture from DNA fragments to be separated by gel electrophoresis and then fixed onto a nitrocellulose filter. The presence of probe sequences can be identified by reacting the filter under hybridization conditions with one or more labeled probles. This method can be used to identify fragments of restriction-enzyme DNA that contain a particular probe sequence and to analyze restriction-fragment length polymorphisms.

Another method to detect the presence of sequences or sequences within a polynucleotide samples is selective amplification by polymerase chain reactions. U.S. Pat. No. 4,683,202 to Mullis, et al. and R. K. Saiki, et al., Science 230:1350 (1985). This method uses primers that are complementary to the opposite ends of the sequence to promote thermal cycling and subsequent rounds of primer-initiated reproduction. There are many ways to identify the amplified sequence. This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample.

More recently methods for identifying known target sequences using probe ligation techniques have been reported. U.S. Pat. No. 4,883,750 to N. M. Whiteley, et al., D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren, et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem. 37:1522 (1991). One approach is called oligonucleotide-ligation assay (?OLA). Two probes or probe elements that span the target area of interest are combined with each other. The probe elements that match the target bases at their confronting ends can be joined using ligation (e.g. by treatment with Ligase). Assemble the ligated probe element and you will see the target sequence.

In a modified version of this approach, the probe elements that have been ligated act as a template to create a pair or complementary probe elements. The target sequence is amplified geometrically by continuous cycles of hybridization, denaturation, and ligation with the two complementary pairs. This allows very small amounts to be detected and/or amplified. This is called ligase-chain reaction (?LCR?) F. Barany.?Genetic Disease Detection Using Cloned Thermostable ligase? Proc. Nat’l Acad. Sci. Sci. PCR Methods & Applications, 1:5-16 (1991).

U.S. Pat. reveals “Another scheme to multiplex detect nucleic acid sequence variations differences” No. Grossman et. al. Sequence-specific probes with a distinguishable label and a distinct ratio of charge/translational frictional drag can be combined to form a target and ligated together. Grossman et. al. used this technique. Grossman et. al., “High-density Multiplex Identification of Nucleic Acid Sequences : Oligonucleotide Ligation Test and Sequence-coded Separation”,? Nucl. Acids Res. 22(21),:4527-34 (1994), for large-scale multiplex analysis of cystic fibrosis transmembrane regulatory gene.

Jou, et. al., ?Deletion Detection in Dystrophin Gene by Multiplex Gap Ligase Chain Reaction and Immunochromatographic Strip Technology,? Human Mutation 5 :86-93 (1995), refers to the use a so-called?gap Ligase Chain Reaction? process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon.

In the field of genetic screening there is a growing demand for methods that can detect the presence or absence in each sequence in a target polynucleotide. There have been 400 mutations associated with cystic Fibrosis. It is important to screen for genetic predispositions to this disease. To make sure that a positive diagnosis of cystic fibrosis can be made, it is best to test every possible gene sequence mutation in the subject’s genome. It would be ideal to test all possible mutation sites using one assay. The prior-art methods are not easily adaptable to detect multiple sequences in an automated, single-assay format.

Solid-phase hybridization assays need multiple liquid-handling steps. Incubation and wash temperatures should be controlled carefully to maintain the stringency required for single-nucleotide mismatch discrimination. This approach is difficult to multiplex, as the optimal hybridization conditions can vary widely between probe sequences.

Allele-specific products PCR products are generally the same size. A given amplification tube can be scored by the presence of or absence the product band in each gel lane. Gibbs et al., Nucleic Acids Res., 17:2437-2448 (1989). This method involves splitting the test sample into multiple tubes using different primer combinations. This increases the assay cost. Different fluorescent dyes can be attached to different allelic primers using PCR. This allows for discrimination of alleles (F. F. Chehab et al. Proc. Natl. Acad. Sci. Sci. It is possible to use electrophoretic mobility to distinguish allelic PCR products from bases modified with bulky side chain. However, this method is constrained by the success of polymerase in incorporating these modified bases and the ability of electrophoresis for large PCR products that differ by one of these groups. Livak et al., Nucleic Acids Res., 20:4831-4837 (1989). Each PCR product can only be used to search for a single mutation. Multiplexing is difficult because of this limitation.

Ligation for allele-specific probes has generally used solid-phase capture” (U. Landegren and al. Science, 241-1077-1080 (1988); Nickerson and al. Proc. Natl. Acad. Sci. USA, 87.8923-8927 (1990), or size-dependent seperation (D. Y. Wu et al. Genomics, 4;560-569 (1989), and F. Barany Proc. Natl. Acad. Sci., 88.189-193 (1991). To resolve allelic signals, Sci., 88.189-193 (1991). This latter method is limited in multiplex scale due to the small size range of ligation probes. A polymerase extension is required for the gap ligase reaction process. The use probes with different ratios of charge/translational drag technique to a complex multiplex will require either a longer electrophoresis time or an alternative method of detection.

There is still a need for a single rapid assay format that can detect the presence of selected sequences in a polynucleotide specimen.

Use Oligonucleotide arrays for nucleic acid analysis

For sequencing, sorting and manipulating DNA, ordered arrays of oligonucleotides have been proposed. The theory behind hybridization of single-stranded DNA molecules cloned to any number of oligonucleotide probes can identify the complementary DNA segments in the molecule. Each oligonucleotide probe in such an array is immobilized on a solid supporting at a predetermined position. With such an array, you can survey all the oligonucleotide fragments in a DNA molecule.

U.S. Pat. 102/030 discloses a method for sequencing DNA molecules using arrays made of oligonucleotides. No. No. al. This involves attaching target DNA to a solid base to which multiple oligonucleotides have been attached. The sequences are created by hybridizing segments of target DNA with oligonucleotides, and then assembling overlapping segments hybridized oligonucleotides. Although the array uses all oligonucleotides that are between 11 and 20 nucleotides in length, there is not much information on how it is made. A. B. Chetverin, et. al.,?Sequencing of Pools of Nucleic Acids On Oligonucleotide Arrays? BioSystems 30, 215-31 (1993);WO 92/16655 Khrapko et. al. ; Kuznetsova, et., DNA Sequencing with Hybridization using Oligonucleotides immobilized in Gel. Chemical Ligation as a Way to Expand the Prospects of the Method, Mol. Biol. 28(20): 290-99(1994); M. A. Livits, et. al.,?Dissociation Of Duplexes Formed By Hybridization DNA with Gel-Immobilized Oligonucleotides J. Biomolec. Struct. & Dynam. 11(4): 783-812 (1994).

Southern’s WO 89/10977 discloses the use a support that contains an array of oligonucleotides that can undergo a hybridization reaction to analyze a nucleic acids sample for known point mutations. It is also capable of linking analysis, genomic fingerprinting, linkage analyses, and sequence determination. You can form the matrix by placing nucleotide base in a chosen pattern on the support. This indicates that the support can be provided with a hydroxyl group. The oligonucleotides are assembled using a pen plotter, or masking.

WO 94/11530 To Cantor also refers to the use an oligonucleotideArray to perform a process called sequencing by hybridization. Oligonucleotides refer to duplexes with overhanging ends, to which target nucleic acid bind and then ligated to non-overhanging portions of the duplex. Streptavidin-coated filter papers capture biotinylated Oligonucleotides before attachment.

WO 93/17126 is used by Chetverin to sort and survey nucleic acid. These arrays contain a constant nucleotide and a variable nucleotide sequence that are both attached to a solid support with a covalent linking moiety. To allow PCR to amplify hybridized strands, the constant nucleotide sequence includes a priming area. The variable region is used to hybridize the strands. These binary arrays can be used for sequencing, sorting, and manipulating fragmented DNA. One embodiment has enhanced sensitivity because the immobilized Oligonucleotide contains a shorter complementary region that is hybridized to it. This leaves some of the oligonucleotide ununcovered. The array is then subjected a hybridization condition so that a complementary nuclear acid annexes to the immobilized Oligonucleotide. The shorter complementary region is joined to the array using DNA ligase. It is not clear how to prepare arrays of oligonucleotides.

WO 92/10588 Fodor et. Fodor et. al. disclose a method for sequencing, fingerprinting and mapping nucleic acid by hybridization to an array oligonucleotides. An immobilized polymer synthesis on a large scale allows for the production of many different oligonucleotides. The substrate surface is functionalized, and a linker group is added to allow oligonucleotides to be assembled on it. Protective groups are found on the substrate and individual nucleotide units in regions where oligonucleotides have been attached. These are selectively activated. This involves using light to image the array, with a mask of different configurations so that exposed areas are deprotected. Deprotected areas undergo a chemical reaction using a protected nucleotide in order to expand the sequence of oligonucleotides being imaged. Binary masking can be used to create multiple arrays at once. The detection of hybridization involves the positional localization. U.S. Pat. Nos. Fodor et. al., U.S. Pat. Nos. Pirrung, et. al., WO90/15070 Pirrung, et. al., A. C. Pease, et. Al., A. C. Pease, et. Natl. Acad. Sci USA 91: 5022-26 (1994). K. L. Beattie, et. K. L. Beattie, et. al., Advances in Genosensor Research. Clin. Chem. 41(5): 700-09 (1995), discloses the attachment of oligonucleotide probes previously assembled to a solid support.

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