Invented by Francis Barany, Monib Zirvi, Norman P. Gerry, Reyna Favis, Richard Kliman, Cornell Research Foundation Inc

The market for Method for designing an addressable array that can detect nucleic acid sequence variations using the ligase detection is a rapidly growing market. This method is used to detect variations in nucleic acid sequences, which are important in many fields, including medical research, genetic testing, and forensic science. The ligase detection method involves the use of a ligase enzyme, which can join two pieces of DNA together. This method is used to detect variations in nucleic acid sequences by detecting the presence or absence of a specific sequence. The method is highly sensitive and specific, making it ideal for detecting even small variations in nucleic acid sequences. The market for this method is driven by the increasing demand for genetic testing and personalized medicine. Genetic testing is becoming more common as people seek to understand their risk for certain diseases and conditions. The ligase detection method is particularly useful in genetic testing because it can detect even small variations in nucleic acid sequences that may be associated with disease risk. In addition to genetic testing, the ligase detection method is also used in medical research. Researchers use this method to study the genetic basis of diseases and to develop new treatments. The method is also used in forensic science to identify individuals based on their DNA. The market for the method for designing an addressable array that can detect nucleic acid sequence variations using the ligase detection is expected to continue to grow in the coming years. As genetic testing becomes more common and personalized medicine becomes more prevalent, the demand for this method is likely to increase. In addition, as researchers continue to study the genetic basis of diseases and develop new treatments, the ligase detection method will continue to be an important tool in their work. Overall, the market for the method for designing an addressable array that can detect nucleic acid sequence variations using the ligase detection is a promising and rapidly growing market. As the demand for genetic testing and personalized medicine continues to increase, this method is likely to become even more important in the years to come.

The Cornell Research Foundation Inc invention works as follows

The present invention relates to a method for designing multiple capture oligonucleotide probles that can be used on a support. These probes will mix with minimal mismatch and the plural capture probes have melting temperature within a narrow range. Further, the present invention relates to an OligonucleotideArray that includes a support with the plurality oligonucleotide probles immobilized on it, a method for using the support to detect single base changes, insertions deletions or translocations in a plurality target nucleotide sequences, as well as a kit to do such detection which includes the support where the oligonucleotides were immobilized.

Background for Method for designing an addressable array that can detect nucleic acid sequence variations using the ligase detection process

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 modification to this approach, the ligated probe element acts as a template for two complementary probe elements. The target sequence can be amplified exponentially by continuous cycles of hybridization, denaturation, and ligation with the two complementary probe elements. 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, in 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. This is used for analysis of nucleic acids samples for genomic fingerprinting, linkage analyses, sequence determination, and genomic fingerprinting. You can create the matrix by placing nucleotide base in a specific 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 large numbers of 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: 5022-25 (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

The sequencing by hybridization of such arrays has many disadvantages. First, it is necessary to synthesize a large number of Oligonucleotides. Second, it is difficult to discriminate between properly hybridized, correctly matched duplexes, and those that are mismatched. Certain oligonucleotides may be more difficult to hybridize under standard conditions. These oligonucleotides can only be identified through extensive hybridization studies.

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