WO2002079518A1

WO2002079518A1 - A method for genotyping individuals for multiple snps

Info

Publication number: WO2002079518A1
Application number: PCT/US2002/009687
Authority: WO
Inventors: Dominic Spinella; Margaret Galvin
Original assignee: Chugai Seiyaku Kabushiki Kaisha
Priority date: 2001-03-29
Filing date: 2002-03-27
Publication date: 2002-10-10

Abstract

This invention provides a method for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid, a method for genotyping individuals for a single nucleotide polymorphism (SNP) of interest, a kit for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid, and an oligonucleotide having an allele specific nucleotide at the 3' end, the 5' end affixed to a solid phase matrix and a sequence complementary to a sequence in a sample nucleic acid.

Description

SPECIFICATION

A METHOD FOR GENOTYPING INDIVIDUALS FOR MULTIPLE SNPs

FIELD OF THE INVENTION

This invention relates to a method for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid, a method for genotyping individuals for a single nucleotide polymorphism (SNP) of interest, a kit for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid, and an oligonucleotide having an allele specific nucleotide at the 3' end, the 5' end affixed to a solid phase matrix and a sequence complementary to a sequence in a sample nucleic acid. BACKGROUND OF THE INVENTION

The determination of a draft nucleotide sequence of the human genome by private and public organizations (Venter, J.C. et al; Science 291 :1305-1351 , 2001 ; Lander et al.; Nature 409: 860-921 ; 2001) has led, as one consequence, to an appreciation of the variation among individuals in the species. Inherited differences in the genome sequence of individuals contribute to phenotypic variation and influence not only physical appearance, but susceptibility to disease, responsiveness to drugs, and numerous other complex traits. Classical genetics follows the co-inheritance of such phenotypic traits with known genotypic variations in order to establish linkage between genotype and phenotype. By far the most common of the genotypic variations in humans is the single nucleotide polymorphism, or SNP.

SNPs are sites within the genome at which more than one of the four canonical base pairs is observed within various members of the population. With the identification of more than 1.4 million of these polymorphic sites scattered throughout the ~3 billion base pairs of human genomic DNA (Weisman et al, Nature 409: 928-933, 2001), a dense map of genotypic markers is now available to facilitate genetic linkage studies and to map the association of even complex phenotypes with the underlying genetic variations that cause them. The genetic analysis of complex phenotypes however potentially requires the determination of the individual sequence variants (genotyping) across hundreds of thousands of SNPs and hundreds to thousands of individuals. While numerous technologies are available for determining a genotype at any individual location in the genome (locus), the simultaneous determination of genotypes across tens of thousands of loci is at best an expensive and laborious undertaking.

This is largely because virtually all technologies for SNP genotyping currently available require the a priori amplification of a segment of DNA containing the locus of interest using techniques such as Polymerase Chain Reaction (PCR). Total genomic DNA is a highly complex mixture and the concentration of any individual sequence within it is extremely low (~100,000 copies per μg genomic DNA); hence, it is not possible to directly analyze SNPs without some method to amplify the signal.

While it is possible to automate the amplification and scoring process using robotics and miniaturization to perform thousands of PCR or other amplification reactions, this need to pre-amplify DNA prior to genotyping is a major limitation which largely prevents genotyping at the scale required to analyze complex phenotypes such as disease susceptibility or drug responsiveness. Moreover, the need to pre-amplify particular segments of DNA requires investigators to guess in advance at which polymorphic loci may be involved in the phenotype under study, rather than assess co-inheritance with all known loci.

What is required to make efficient use of the accumulating variation data in the human genome is a method in which tens to hundreds of thousands of SNPs can be simultaneously and rapidly analyzed in a cost-effective manner across multiple patient populations. SUMMARY OF THE INVENTION

This invention relates to a method for identifying a specific nucleotide at a defined site, such as a SNP, of a sample nucleic acid that is dependent on the correct base pairing of an oligonucleotide with the sample nucleic acid and an enzyme having a nucleic acid polymerization activity.

In one aspect, this invention relates to a method for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid. In one aspect, this invention also relates to a method for genotyping individuals for a SNP of interest in a genomic DNA fragment based on the above method.

In one aspect, this invention also relates to a kit for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid.

In one aspect, this invention also relates to an oligonucleotide having an allele specific nucleotide at the 3' end, the 5' end affixed to a solid phase matrix and a sequence complementary to a sequence in a sample nucleic acid.

This invention is used to characterize one or more nucleotides in biological samples and is useful in the diagnosis of diseases, the testing of susceptibility to disease and responsiveness to drugs, the identification of individuals and their parentage, and others. BRIEF DESCRIPTION OF DRAWINGS

Figure 1 demonstrates allele specific fluorescent oligonucleotide extension on a slide using 15 ng of β2 microglobulin PCR product as template and Streptavidin-Allophycocyanin (SA-APC) as method for detection.

Figure 2 demonstrates allele specific fluorescent oligonucleotide extension on a slide using 0.04 ng β2 microglobulin PCR product as template and Tyramide Signal Amplification (TSA) as method for detection. DETAILED DESCRIPTION OF THE INVENTION

The detailed description of the invention which follows is not intended to be exhaustive or to limit the invention to the precise details or examples disclosed. Details and examples have been chosen to explain the invention to others skilled in the art. The processes of this invention described herein and in the claims, may be performed in several ways. Preferred methodologies are described as follows.

As used above, and throughout the description of the invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings. "An allele specific nucleotide", as used herein, means a nucleotide of an oligonucleotide corresponding to a specific nucleotide at the defined site of a sample nucleic acid. The allele specific nucleotide may form a base pair to the specific nucleotide at the defined site. The allele specific nucleotide shows a single nucleotide polymorphism and locates at the 3' end of the oligonucleotide. A set of oligonucleotides has a variety of nucleotides in a position of the allele specific nucleotide that can be two, three or four nucleotides. "Annealing", as used herein, means forming the hydrogen bond between an oligonucleotide or an oligonucleotide extension product and a sample nucleic acid. After dissociating step, an oligonucleotide or an oligonucleotide extension product and a sample nucleic acid are allowed to re-anneal by lowering the temperature. Annealed nucleic acids may be subject to a polymerization agent to extend a chain of an oligonucleotide or an oligonucleotide extension product.

"A detectable label", as used herein, means any label used for nucleotides of four kinds of dNTP to give a signal that can be detected by conventional means. An amount of the signal of the label is qualitatively and quantitatively estimated and is compared in different oligonucleotides. Several approaches can be used for preparing the detectable label, (1) radioactivity and its detection by either autoradiography or scintillation counting, (2) fluorescence or absorption spectroscopy, (3) mass spectrometry, or (4) enzyme activity, using a protein moiety, but not limited to these. Examples of the detectable label include biotin, fluorescent materials (including chelatable metals such as the lanthanides, organic molecules, ultra small or quantum confined materials, and precursors thereof), radioisotope, molecules which provide resonance properties which may be detected, and the like. Such labels may be made to be amplified as an optional step in the reaction. For example, the interaction of biotin and streptavidin may optionally be used to enhance or amplify the fluorescence signal. Examples of nucleotides with detectable labels include a biotinylated nucleotide, fluorescently labeled nucleotide, radio labeled nucleotide, modified nucleotide with altered mass. The nucleotide with detectable label can be prepared by any known methods. When biotin is used as a detectable label, a secondary method for detection is necessary that adopts streptavidin. Two secondary methods include Streptavidin-fluorescent dye conjugate and Tyramide signal Amplification (US Patent 5,196,306, US Patent 5,731 ,158). Preferably such labels are detectable in the subfemptomolar range. Signal intensity of the total amount of the detectable label can be measured and compared.

"Dissociating" as used herein, means separating a primer (an oligonucleotide) from a template (a sample nucleic acid) that forms a duplex. An optimal condition is used for dissociation, for example, raising the temperature of mixture solution by heating up. Hydrogen bond formed between adenine and thymine, and guanine and cytosine in between nucleotides in the oligonucleotide and sample nucleic acid becomes unstable at a temperature over a melting point and is dissociated. Dissociated nucleic acids are not double stranded and are kept single-stranded. "A duplex" as used herein, means hybridized nucleic acids comprising an oligonucleotide and a sample nucleic acid. The oligonucleotide and the sample nucleic acid form the hydrogen bond to provide a hybridized oligonucleotide. By a polymerization agent, the duplex incorporates nucleotides with detectable label.

"Hybridizing", as used herein, means forming a hydrogen bond between an oligonucleotide and a sample nucleic acid. The oligonucleotide hybridizes with its complementary chain of a sample nucleic acid that gives a hybridized oligonucleotide. Hybridization condition preferably accompanies stringency for the annealing of nucleic acids. It is preferred that conditions are chosen to optimize optimal hybridization of the oligonucleotides to the sample nucleic acid of interest. Hybridization is done under an optimal condition.

"An identity", as used herein, means the kind of a residue of a nucleotide having a base selected from adenine, guanine, cytosine or thymine. When a sample nucleic acid contains a specific nucleotide at the defined site, the identity of the specific nucleotide is determined by examining its base-pairing nucleotide. "A nucleotide derivative", as used herein, means, means a nucleotide in which any of the natural structures have been altered or modified. The nucleotide derivatives include modifications to the phosphodiester linkages, the sugars (ribose in the case of RNA or deoxyribose in the case of DNA) and/or the purine or pyrimidine bases into a nucleotide. Modified phosphodiester linkages include phosphorothioates, phosphotriesters, methylphosphonates and phosphorodithioates.

Preferably such modified linkages block all exonuclease activity. Such linkages are preferred where polymerization agents which have exonuclease activity are used. "An oligonucleotide", as used herein, means an nucleic acid, an oligoribonucleotide, or a copolymer or polymer of deoxyribonucleotides and ribonucleotides that are designed to have a sequence complementary to a sequence in a sample nucleic acid or a segment of a sample nucleic acid. The oligonucleotide functions as a primer to extend the nucleic acid chain, and has an allele specific nucleotide, that may be corresponding to a SNP, at its 3' end. The oligonucleotide has preferably the 5' end affixed covalently or by other means to a surface of a solid phase matrix, such as a glass surface. The length of the sequence of the oligonucleotide that is complementary to the sequence in the sample nucleic acids preferably ranges form approximately 10-50 nucleotides. The oligonucleotide can contain not only nucleic acids but also other components such as a nucleotide derivative, a spacer as long as the oligonucleotide is able to hybridize with a sample nucleic acid. Examples of other components include phosphorothioate, biotin, and amine. The oligonucleotides having a nucleotide corresponding to all the allelic variants, that are usually two, but conceptually three or as many as four, of any individual SNP are prepared and are used as a set of oligonucleotides. The oligonucleotide may be provided as an oligonucleotide composition. The oligonucleotides can be prepared by any known conventional methods.

"An oligonucleotide extension product", as used herein, means a primer extension product that is an oligonucleotide extended by a polymerization agent using a sample nucleic acid as a template DNA. The Oligonucleotide extension product may hybridize with the sample nucleic acid. By the extension of the oligonucleotide, incorporation of labeled nucleotides makes it detectable.

"An optimal condition" as used herein, means conditions in which stable hybridization of complementary oligonucleotides is maintained, but mismatches are not (Sambrook et al., Molecular Cloning (1989), see for example, 11.46; RNA hybrids and 9.51 , RNA: DNA hybrids). Preferably, optimal condition means optimization of temperature, charge, salt concentration, template and oligonucleotide concentration as well as addition of co-solvents such as glycerol, DMSO, betaine that lower the melting temperature of the matched base pairs.

"A polymerization agent", as used herein, means an enzyme having a polymerization activity of a nucleic acid to extend an oligonucleotide along a template nucleic acid of a sample nucleic acid, and having minimal or no 3'-5' exonuclease function. The polymerization agent is preferably 3'-5' exonuclease minus (exo ) polymerase. The polymerization agent is preferably thermostable so that it maintains the activity of the nucleotide polymerization by a repetitious procedure of dissociating and annealing that varies the temperature. While error rate of the polymerization agent is not crucial, it is preferred that the polymerization agent has 3' specificity to avoid extension of mismatches. As it relates to mismatches, it is also preferred that the polymerization has little, more preferably no exonuclease activity, to avoid alteration (replacement of bases) of the oligonucleotide. The polymerization agent preferably has a nucleotide extension rate of at least 30 bases/second, preferably 1000 bases/second. The polymerization agent is included into an oligonucleotide (a primer) extension reagent with other reagents and solution upon use. The conditions for the oligonucleotide extension reaction can be created, in part, by the presence of a suitable template-dependent polymerization agent. Examples of the polymerization agent include DNA and RNA polymerases. The DNA polymerase can be of several types. The DNA polymerase must, however, be primer (the oligonucleotide) and template (a sample nucleic acid) dependent. For example, E. coli DNA polymerase I or the "Klenow fragment" thereof, T4 DNA polymerase, T7 DNA polymerase ("Sequenase") and its derivatives, T. aquaticus DNA polymerase and its derivatives, a retroviral reverse transcriptase, P. fu osis DNA polymerase, or Vent polymerase can be used. For RNA, RNA polymerases such as T3 or T7 RNA polymerase are useful in appropriate protocols. Conditions appropriate for the polymerase must be used, and different temperature ranges are used for hybridization and extension reactions. Polymerases can be isolated from a natural source or commercially available ones can be used.

"A sample nucleic acid", as used herein, means any nucleic acid comprising a specific nucleotide at the defined site or position, the identity of which is to be determined. The sample nucleic acid is used as a template in extending nucleic acid chain of an oligonucleotide. The sample nucleic acid may contain one or more specific nucleotide(s) at the defined site or position which can be a SNP(s). The sample nucleic acid can be prepared from DNA or RNA, genomic DNA or cDNA, RNA transcripts thereof, or cDNA prepared from RNA transcripts thereof, preferably, a genomic DNA fragment. The sample nucleic acid can also comprise extragenomic

DNA from an organism. Also, the sample nucleic acid can be synthesized by the polymerase chain reaction. The sample nucleic acid can be taken from any organism, or in another embodiment may be manufactured. Some examples of organisms to which the method of this invention is applicable include plants, microorganisms, viruses, birds, vertebrates, invertebrates, mammals, such as humans, primates, horses, dogs, cows, cats, pigs, sheep, etc. The sample nucleic acid are preferably sheared and melted prior to be hybridized with the oligonucleotide after the isolation from a source of interest. "A single nucleotide polymorphism (SNP)", as used herein, means a site within the genome at which more than one of the four canonical base pairs is observed within various members of the population. One or more SNPs can be included in a sample nucleic acid. SNPs may be related to susceptibility to disease and responsiveness to drugs, and is used in the diagnosis of diseases and the identification of individuals and their parentage, and so on.

"A solid phase matrix", as used herein, means any solid form of material or a substance that is used to affix an oligonucleotide or a set of oligonucleotides on it. The solid phase matrix is preferably inert to the reagents and reaction, as well as being substantially insoluble in the media. For example, material of the solid phase matrix can be glass, plastic, metal or silicon. Preferably the matrix is designed to avoid nonspecific binding of reagents, including polymerization agents, oligonucleotides, nucleic acids and the like, while allowing for permanent attachment of the oligonucleotide. As such it may be preferred that the matrix have sufficient hydrophobicity or hydrophilicity to avoid such interactions. Representative solid phase matrix includes a glass slide, a plastic bead, or silicon based chip. The solid phase matrix can be prepared by known conventional methods, and can be of any numbers of configurations.

"A spacer", as used herein, means a molecule or a group of molecules that connects two or more molecules, such as a nucleotide, a nucleotide derivative, a solid phase matrix, or other spacers. The spacer serves to place the two or more molecules in a preferred configuration. Hence, an oligonucleotide or a set of oligonucleotides is/are able to hybridize with a sample nucleic acid with minimal steric hindrance from the oligonucleotide or the set of oligonucleotides, or the solid phase matrix.

"A specific nucleotide", as used herein, means a nucleotide at the defined site to be identified. The specific nucleotide is in a sample nucleic acid and corresponds to a nucleotide at the 3' end of the oligonucleotide that is an allele specific nucleotide. The specific nucleotide can be corresponding to a SNP.

"A spot", as used herein, means a defined area that has the same type of oligonucleotide is attached. When extension of oligonucleotide happens, nucleotides with detectable label are incorporated into the oligonucleotide chains. The detectable label emits signal in a spot where allele specific oligonucleotides are attached to solid phase matrix and then the signal of the spot is detected by known methods. The signal intensity of the spot is qualitatively or quantitatively determined. Methods of preparing the spot include control of evaporation rate if the oligonucleotide is applied by a solvent such as water, control of the amount of oligonucleotide employed per spot, density of spots on the solid phase matrix. Many such methods are known in the art.

The following abbreviations have the indicated meanings:

AmC6 Amine label with C6 spacer diH₂0 Deionized, Purified Water DMSO Dimethyl sulfoxide

HLA Human Leukocyte Antigen

HRP Horse Radish Peroxidase

PCR Polymerase Chain Reaction

SA-APC Streptavidin-Allophycocyanin SDS Sodium Dodecyl Sulfate

SSC Sodium Chloride, Sodium Citrate

TSA Tyramide Signal Amplification

TNB Tris; Sodium Chloride; Blocking Reagent

TNT Tris; Sodium Chloride; Tween 20 X Times

The inventors intend to specifically include in this invention other available methods of enhancing hybridization, annealing, dissociating and the like. Such methods include methods of increasing or decreasing local concentrations of nucleic acids, utilizing the charge of a nucleic acid to enhance hybridization by applying a positive or negative charge to the solid phase matrix or using a charged matrix, and any other methods to alter kinetics or concentration of the reagents. It is recognized by the skilled artisan that reagents can be used as premixes, or all present in the initial mixture before the method or process begins. Furthermore, steps within the method are not affected by the fact that reagents may be present to be used in a subsequent step. In addition, the order of steps in the method may be reordered. Moreover, the order of addition of reagents does not affect the method or process either, though the skilled artisan will recognize that the process may optimally have a preferred order of addition.

This invention provides a method for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid comprising the steps of: (a) providing an oligonucleotide having an allele specific nucleotide at the 3' end and a nucleotide sequence complementary to a sequence in said sample nucleic acid in a single stranded form,

(b) hybridizing said sample nucleic acid with said oligonucleotide, thereby said allele specific nucleotide base-pairs to said specific nucleotide, to provide a hybridized oligonucleotide,

(c) exposing said hybridized oligonucleotide to a polymerization agent in a mixture containing nucleotides having a detectable label, under a condition that said polymerization agent allows said hybridized oligonucleotide having base-paired nucleotides to extend the oligonucleotide by incorporating said nucleotides having a detectable label to form a detectable oligonucleotide extension product, and

(d) detecting the presence or absence of the detectable label of said detectable primer extension product.

A sample nucleic acid containing a sequence of interest is treated, if such a sample nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position. If the nucleic acid of interest is single-stranded, this step is not necessary. The sample nucleic acid included can be obtained from any sources, such as human, mammalian, etc. A specific nucleotide at the defined site may be a SNP and the SNP could be detected by this method. The sample nucleic acid can be free from or preferably affixed to a solid phase matrix via a bond, for example covalent bond. The stabilization of the sample nucleic acid to the solid phase matrix is made by any conventional means. The sample nucleic acid is contacted with an oligonucleotide under hybridizing conditions. The oligonucleotide has a sequence complementary to a sequence in the sample nucleic acid such that it is capable of hybridizing with a stretch of nucleotide bases present in the sample nucleic acid, including the specific nucleotide base to be identified. The oligonucleotide and the sample nucleic acid form a duplex and the sample nucleic acid can be a template nucleic acid for oligonucleotide extension.

The oligonucleotide is hybridized with the sample nucleic acid having the specific nucleotide which is complementary to it. Accordingly depending on a sequence of the sample nucleic acid of interest, the sequence of the oligonucleotide can be designed and can be changed. The sequence included in the oligonucleotide is not limited to ones described in this specification. Sequences included in an oligonucleotide are designed to include any SNPs of interest. Exempts of SNPs of interest are described in Nature 15 February 2001 , v409, 928 - 933.

The duplex between the oligonucleotide and the sample nucleic acid includes the specific nucleotide base to be identified which is paired with the allele specific nucleotide at the 3' end of the oligonucleotide. The sample nucleic acid is contacted with the oligonucleotide under conditions permitting forming base-pairing of the complementary oligonucleotide present in a mixture with the specific nucleotide base to be identified. If a sample nucleic acid has a different nucleotide from the specific one at the defined site, the oligonucleotide could hybridize with the nucleic acid polymer, however, polymerization would not likely occur.

Enzymatic oligonucleotide extension of the oligonucleotide in the resultant duplex is performed by exposing the hybridized oligonucleotide to a polymerization agent. A suitable condition can be chosen for the polymerizing reaction. The enzymatic oligonucleotide extension with labeled nucleotides, catalyzed by a polymerization agent, for example, DNA polymerase, thus depends on correct base- pairing of the nucleotide at the 3' end of the oligonucleotide and the specific nucleotide at the defined site in a sample nucleic acid. Since a polymerization agent has minimal or no exonuclease function (exo minus), the polymerization agent does not unlikely chew back the incorrect nucleotide that does not base pair and therefore an oligonucleotide extension reaction could not likely occur. Oligonucleotide extension products incorporate nucleotides having detectable label. Detecting the presence or absence of the detectable label is accomplished by conventional means depending on the kind of detectable label. When the oligonucleotide extension occurs and the presence of the detectable label is confirmed the sample nucleic acid possesses a specific nucleic acid at the defined site. Part or all of one type of the nucleotides to be incorporated in the oligonucleotide extension products may be labeled and one, two, three or four nucleotides may be labeled.

The signal of the detectable label in a spot in which the sequence of the oligonucleotide is entirely matched to the template of the sample nucleic acid exceeds the signal in a spot containing a 3' end mismatched oligonucleotides. The detectable label incorporated in the oligonucleotide extension product can be compared between a group that possesses the specific nucleotide at the defined site and a group that does not possess the one. Consequently, the excessive amount of the detectable label shows the existence of the specific nucleotide at the defined site.

Oligonucleotide extension reaction can be repeated to enhance the incorporation of a signal of the labels and the signal intensity. After exposing said hybridized oligonucleotide to a polymerization agent in a mixture containing nucleotides having a detectable label, two steps can be further taken including (i) a dissociating step of said oligonucleotide extension product from the sample nucleic acid and then (ii) an annealing step of the sample nucleic acid on said oligonucleotide extension product. These steps are repeatedly performed in the presence of said polymerization agent to facilitate the oligonucleotide extension reaction of the oligonucleotide having an allele specific nucleotide at the 3' end and the incorporation of labeled nucleotides in a primer extension product.

Detecting the presence or absence of the detectable label of the detectable oligonucleotide extension product can be done by measuring a total amount of the signal of the detectable label. Detection of the label accompanies comparing signal intensity of each spot by a scanner.

Based on the method above, this invention also provides a method for genotyping individuals for a SNP of interest in a genomic DNA fragment comprising (a) providing a set of oligonucleotides having allele specific nucleotides at the 3' end corresponding to said SNP and a nucleotide sequence complementary to a sequence in said genomic DNA fragment in a single stranded form,

(b) hybridizing said genomic DNA fragment with said set of oligonucleotides, thereby at least one kind of said allele specific nucleotides base-pairs to a nucleotide corresponding to said SNP, to provide hybridized oligonucleotides,

(c) exposing said hybridized oligonucleotides to a polymerization agent in a mixture containing nucleotides having a detectable label, under a condition that said polymerization agent allows said hybridized oligonucleotides having base-paired nucleotides to extend the oligonucleotides by incorporating said nucleotides having a detectable label to form detectable oligonucleotide extension products,

(d) dissociating said genomic DNA fragment from said detectable oligonucleotide extension products,

(e) annealing said genomic DNA fragment to said detectable oligonucleotide extension products, (f) repeating said dissociating step of (d) and said annealing step of (e) in the presence of said polymerization agent, thereby allowing said oligonucleotides having base-paired nucleotides to extend the oligonucleotides by incorporating said nucleotides having a detectable label, (g) detecting the presence or absence of said detectable label of the detectable oligonucleotide extension products, and

(h) comparing signal intensity of said detectable label of the detectable oligonucleotide extension products.

An unamplified genomic DNA fragment is isolated from any individual using standard methods known in the art, sheared and melted by standard techniques, and is hybridized to a set of oligonucleotides that are affixed to a solid phase matrix. The set of oligonucleotides can contain an allele specific nucleotide at the 3' end and the allele specific nucleotide is a variety of nucleotide corresponding to a SNP. Loci containing a specific nucleotide at the defined site of a genomic DNA fragment are allowed to base-pair to an allele specific nucleotides of the oligonucleotides. The oligonucleotides are then allowed to extend by copying the template genomic DNA fragment using a polymerization agent such as a DNA polymerase enzyme which lack 3'-5' exonuclease activity. Such enzymes are inefficient in extending oligonucleotides in which the 3' nucleotide base is mismatched to the template strand.

Hence, if spots contain oligonucleotides having matched and mismatched nucleotide that define alternative alleles of a bi-allelic SNP, the oligonucleotide extension reactions will proceed in the region containing the matched oligonucleotide primer. The oligonucleotide extension reactions are allowed to proceed in the presence of labeled nucleotides, so the labels are incorporated into a chain of the spot on which the entirely matched oligonucleotide is located.

Once oligonucleotide extension reactions have been allowed to proceed for a time, the oligonucleotides are heated to dissociate the template strand of the sample nucleic acid from the oligonucleotides to which they have been annealed. The oligonucleotides are then cooled to a hybridization temperature upon which the template strand of the sample nucleic acids is free to re-anneal to another, oligonucleotide in the region, and the extension process allowed to proceed anew. This process can be repeated to achieve a linear amplification until signal intensity from the labeled nucleotides is enough to be detected.

The signal in the spot in which the oligonucleotide is entirely matched to the template of the sample nucleic acid exceeds the signal in the spot containing a 3' mismatched oligonucleotides. Hence, a signal to noise ratio will be achieved allowing unambiguous assignment of genotype. In the case of a heterozygous individual in which both SNP templates would be present in the DNA sample at equivalent concentrations, both spots would achieve equivalent signal intensity.

The method of this invention can be performed with no pre-amplification of DNA. Sufficient detection sensitivity is attained by in situ linear amplification achieved by thermal cycling the oligonucleotide which allows a single template molecule of the sample nucleic acids to extend multiple oligonucleotides and by the incorporation of multiple molecules of label per extended oligonucleotide. Because many thousands of oligonucleotides corresponding to an equivalent number of SNPs can be spatially arrayed in spots of a solid phase matrix, an arbitrarily large number of oligonucleotide extension reactions can be performed together, allowing simultaneous genotyping at large numbers of loci. The X-Y position of allele discriminating oligonucleotide spots on the solid phase matrix defines any particular locus.

This invention also provides a method of typing a sample of nucleic acids which comprises identifying the base or bases present .at each of one or more specific positions, each such nucleotide base being identified using one of the methods for determining the identity of a nucleotide base at a specific position in a sample nucleic acid of interest. Each specific nucleotide in the sample nucleic acid of interest is determined using a different oligonucleotide. The identity of each nucleotide base or bases at each position can be determined individually or the identities of the nucleotide bases at different positions can be determined simultaneously.

This invention further provides a method for identifying different alleles in a sample containing nucleic acids. This method comprises identifying the base or bases present at each of one or more specific positions. The identity of each nucleotide base is determined by the method for determining the identity of a nucleotide base at a specific nucleotide in a sample nucleic acid of interest.

This invention also provides a method for determining the presence or absence of a particular nucleotide sequence in a sample of nucleic acids. First, a sample nucleic acid is treated, if such sample of nucleic acids contains double- stranded nucleic acids, so as to obtain single-stranded nucleic acids. If the nucleic acids in the sample are single-stranded, this step is not necessary. Second, the sample nucleic acid is contacted with an oligonucleotide under hybridizing conditions. The oligonucleotide is capable of hybridizing with the particular nucleotide sequence, if the particular nucleotide sequence is present, so as to form a duplex between the oligonucleotide and the particular nucleotide sequence. Since an amount of a detectable label is present in any of the oligonucleotide extension products, the oligonucleotide does hybridize to the template of the sample nucleic acid, and, therefore, the particular nucleotide sequence was present in the sample of nucleic acids.

This invention also provides a method for determining the genotype of an organism at one or more particular genetic loci. For example, one may obtain from the organism a sample containing genomic DNA and identify the nucleotide base or bases present at each of one or more specific positions in sample nucleic acids of interest. The identity of each such base is determined by using one of the methods for determining the identity of a nucleotide base at a specific position in a sample nucleic acid of interest. The identities of the nucleotide bases determine the different alleles and, thereby, determine the genotype of the organism at one or more particular genetic loci.

This invention also provides a kit for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid comprising, in a packaged combination,

(a) an oligonucleotide or a set of nucleotides having a sequence of interest, wherein said oligonucleotide(s) is(are) selected so as to be sufficiently complementary to a strand of a sample nucleic acid to anneal therewith,

(b) a polymerization agent, and

(c) nucleotides having a detectable label.

The oligonucleotide, the polymerization agent and the nucleotides of four kinds of_dNTP having a detectable label are provided in a packaged combination, and can be prepared by methods described throughout the specification. The oligonucleotide can be a set of oligonucleotides. The kit could include any other materials or reaction compositions and not limited to ones described here. For example, the kit could further include buffer for preparing solution for reaction. The kit could further include a solid phase matrix for affixing a nucleic acid polymer to it. This kit could contain reagents and protocol to affix oligonucleotides to a solid phase matrix. This kit could also comprise oligonucleotides attached to solid phase matrix. These oligonucleotides would be predetermined to be specific for SNPs of interest. This kit is useful in determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid and genotyping individuals for multiple SNPs. The kit of this invention is useful in determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid. This invention also provides an oligonucleotide for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid having an allele specific nucleotide at the 3' end, the 5' end affixed to a solid phase matrix and a sequence complementary to a sequence in a sample nucleic acid. The allele specific nucleotide corresponding to a SNP can be any known nucleotide and can be related to any diseases such as infectious diseases, genetic disorders, and the identification of individuals and their parentage. The 5' end of the oligonucleotide is affixed to a solid phase matrix. The sequence included in the oligonucleotide may include a sequence complementary to a sequence that is adjacent to the SNPs nucleotide at its 5' end of a sample nucleic acid. The oligonucleotide can be provided as a set of oligonucleotides. The oligonucleotide of this invention is useful in determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid.

The following examples illustrate the invention, and are not intended to limit the invention in any way. It is expected that with the guidance of this specification, and the art that the skilled artisan is fully enabled to use the invention and any variation thereof within the claims. The invention uses known material, many of which are commercially available. EXAMPLES EXAMPLE 1 Allele specific fluorescent oligonucleotide extension using SA-APC (1) Design of the oligonucleotides

The human gene encoding β2-microglobulin (Rosa, F. et al; EMBO J. 2: 239- 243, 1983) is known to possess a polymorphic site in the ATG start codon of the gene. As shown in below, a mutant allele in which the G residue in this codon is changed to C is present in some individuals, which prevents the translation of this protein and the subsequent expression of HLA class I molecules with which the wild-type protein associates on cell surfaces.

ATGTCTCGCTCCGTGGCCTTAGCTGTGCTCGC Normal (SEQ ID No. 1) _ -c Mutant (SEQ ID No. 2) A pair of oligonucleotides B2M_sC and B2M_sG were synthesized which are entirely complementary to a segment either the normal or mutant β2 microglobulin sequence and in which the 3'-most base defines the polymorphism as shown below:

Normal oligonucleotide (B2M_sC)

5'- [AmC6](12T) CACAGCTAAGGCCACGGAGCGAGAC (SEQ ID No. 3)

Mutant oligonucleotide (B2M_sG)

5'- [AmC6](12T) CACAGCTAAGGCCACGGAGCGAGAG (SEQ ID No. 4)

(2) Oligonucleotide attachment to slide

The oligonucleotides were synthesized with 5' amine groups separated from the first (5') base by a 6-carbon spacer molecule followed by a 12 T spacer. There is also a phosphorothioate linkage between the last two bases at the 3' end to alleviate 3' exonuclease activity. The amine-labeled oligonucleotides were then spotted onto commercially available, aldehyde-coated glass slides (Xenopore, Telechem), resulting in the formation of a reversible Schiff base linkage between the slide and the oligonucleotides.

Approximately 0.3 μl of a 10 μM solution of each oligonucleotide in 15 mM sodium phosphate and a spotting solution (1X Array It™, TeleChem) were spotted onto the slides at various positions and allowed to react overnight. The slides were then washed in 0.2% SDS twice for 2 min each and diH₂0 twice for 2 min each, were reduced with 70mM sodium borohydride in 1X PBS pH 7.4 to form an irreversible imine linkage and block any further reactive sites, and then were washed again in 0.2% SDS three times for 1 min each and diH₂0 three times for 1 min each. For the TSA detection example, the 0.2% SDS twice, for 2 min each and diH₂0 twice, for 2 min each wash was eliminated and samples were reduced with sodium borohydride after spotting.

(3) Preparation of the sample nucleic acid

The sample nucleic acid was prepared from human cell lines HEL 92.1.7 (ATCC T1 B-180) and Daudi (ATCC CCL-213) by PCR amplification. A pair of primers used in the amplification designed as forward primer B2M_FB (SEQ ID No. 5) and reverse primer B2M_RB (SEQ ID No. 6) was prepared.

B2M_FB (SEQ ID No. 5) 5'- TCTAACCTGGCACTGCGTCG - 3" B2M_RB (SEQ ID No. 6) 5'- CTCACGCTGGATAGCCTCC - 3'

10 ng of genomic DNA comprising β2 microglobulin gene from Daudi or HEL 92.1.7 was used as a template DNA. PCR reaction was set up in 25 ul reaction volume containing the genomic DNA, 15 pmol of each forward and reverse primer, 200 uM of each dATP, dCTP, dGTP, and dTTP (Perkin Elmer), 1X AmpliTaq Gold Buffer (Perkin Elmer), 5 Unit AmpliTaq Gold (Perkin Elmer).

The PCR amplification was performed under 95C, 10 min, 95C, 30 sec, 55C, 1 min, x 25 cycles, and 72C, 2 min, 4C hold, and a PCR product of 213 bp β2 microglobulin was obtained. The sequence of the 213 bp PCR product is described in the SEQ ID No. 7. The resultant PCR product was purified and was used as the sample nucleic acid in the following experiments.

(4) Oligonucleotide extension reactions

The slides prepared in (2) were dried by a quick centrifugation. 100 μl oligonucleotide extension reactions were set up in sealed hybridization chambers containing 0.04 ng to15 ng sample nucleic acid containing the normal β2 microglobulin G allele, 200 μM each dATP, dCTP, dGTP (Perkin-Elmer), and biotin- 16-dUTP (Roche Molecular Systems), 2mM MgCI₂ (Perkin-Elmer), 1X Stoffel Buffer (Perkin-Elmer), and 20Units AmplTaq DNA Polymerase Stoffel Fragment (Perkin- Elmer).

Slides were placed onto thermal heat plate (Heat Platen, Genetec, Inc.) in a thermocycler (GeneAmp^® 9700 PCR thermocycler, PE Applied Biosystems).

Oligonucleotide extension reagents were added to hybridization chamber at an elevated temperature to reduce bubbles and sealed. Cycling parameters were the following: 95°C, 30 sec; 55°C, 1 min.; 72° C 2 min for 30 cycles.

(5) Detection with Streptavidin-Allophycocyanin (SA-APC) After cycling, excess oligonucleotide extension reagents were washed from slides in a solution containing 2X SSC and 0.1% SDS for 15 min at room temp. Additional washing was done in 0.2X SSC for 5 min and 0.1X SSC for 5 min. Slides were then blocked with a blocking buffer (Superblock^® Blocking Buffer, Pierce) for 15 min at room temperature. SA-APC (Molecular Probes) was diluted to 10 ug/ml in a solution (BlockAid™ solution, Molecular Probes). Diluted SA-APC solution was then placed in spots on slides and non-stick cover slips were layered on top.

Slides were then incubated in a hybridization cassette at 37° C for 30 min. After a final brief wash in a blocking buffer (Superblock^® Blocking Buffer), the slide was viewed using a scanner (ScanArray^® 5000 microarray scanner, GSI Lumonics). Quantitation of signal intensity of the spots was done using software (QuantArray^® software, GSI Lumonics). A measurement of specificity is the ratio of signal intensity between spots containing matched primer-template pairs and those containing mismatched primer template pairs.

The results are shown in Table 1 and Figure 1. When the signal intensity of the spots was measured, there was approximately a 4.3 fold difference in signal intensity between the matched primer-template pairs and the mismatched primer- template pairs. Therefore allele specificity was demonstrated by this experiment.

EXAMPLE 2 Allele Specific Fluorescent Oligonucleotide Extension using TSA Detection

The same procedures (1)-(4) of Example 1 were performed. The sample nucleic acid was a 213 bp PCR product containing the normal human β2 microglobulin G allele as in Example 1. (5) Detection with Tyramide Signal Amplification (TSA™, NEN) Fluorescence System

After cycling, excess oligonucleotide extension reagents were washed from slides by a short wash in diH₂0 at room temp. Slides were then blocked in 300 μl TNB Buffer (0.1M Tris-HCI, pH 7.5; 0.15M NaCI; 0.5% Blocking Reagent from NEN) for 30 min at room temperature. After blocking, the slides were incubated for 30 min at room temp in 300 μl of Streptavidin-HRP reagent (NEN) (1 :50 dilution in TNB Buffer). Slides were then washed in TNT Buffer (0.1 M Tris-HCI, pH 7.5; 0.15M NaCI; 0.05% Tween^®20) 3 times at room temp for 5 minutes each.

After washing in TNT Buffer, the slides were incubated in 300 ul Cyanineδ- Tyramide conjugate (NEN) diluted 1 :50 in a solution (Amplification Diluent, NEN) for 10 minutes. Lastly, the slides were washed three times for 5 minutes in TNT Buffer. For analysis, the slide was viewed using a scanner (ScanArray^® 5000 microarray scanner, GSI Lumonics). Quantitation of signal intensity of the spots was done using software (QuantArray^® software, GSI Lumonics).

The results are shown in Table 2 and Figure 2. When the signal intensity of the spots was measured, there was approximately 5.9 fold difference in signal intensity between the matched primer-template pairs and the mismatched primer- template pairs. Therefore, allele specificity was demonstrated by this experiment.

Table 2

Unless otherwise indicated, the experiments in Examples were made under kit manufacturers' instruction.

This invention involves genomics, manipulations, and protocols that have been, or are amenable to being, automated. Thus incorporation of the preferred mode of practice of this invention into the operation of a suitably programmed robotic workstation is within the skill of the artisan in the field.

Claims

What is claimed:

1. A method for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid comprising the steps of:

(a) providing an oligonucleotide having an allele specific nucleotide at the 3' end and a nucleotide sequence complementary to a sequence in said sample nucleic acid in a single stranded form,

2. The method of claim 1 , wherein said specific nucleotide at the defined sites is a single nucleotide polymorphism (SNP).

3. The method of claim 1 , wherein the 5' end of said oligonucleotide is affixed to a solid phase matrix.

4. The method of claim 3, wherein said oligonucleotide affixed to the solid phase matrix further comprises a component selected from the group consisting of a nucleotide derivative, and a spacer.

5. The method of claim 1 , wherein said sample nucleic acid is derived from unamplified genomic DNA.

6. The method of claim 1 , wherein said polymerization agent is a 3'-5' exonuclease minus DNA polymerase.

7. The method of claim 1 , wherein said detectable label is selected from the group consisting of biotin, fluorescence, and radioisotope.

8. The method of claim 1 , further comprising a dissociating step of said sample nucleic acid from the detectable oligonucleotide extension product and then annealing step of said sample nucleic acid on the detectable oligonucleotide extension product, after the step (c), and repeating said dissociating step and said annealing step in the presence of said polymerization agent.

9. A method for genotyping individuals for a SNP of interest in a genomic DNA fragment comprising:

(a) providing a set of oligonucleotides having allele specific nucleotides at the

3' end corresponding to said SNP and a nucleotide sequence complementary to a sequence in said genomic DNA fragment in a single stranded form,

(e) annealing said genomic DNA fragment to said detectable oligonucleotide extension products,

(f) repeating said dissociating step of (d) and said annealing step of (e) in the presence of said polymerization agent, thereby allowing said oligonucleotides having base-paired nucleotides to extend the oligonucleotides by incorporating said nucleotides having a detectable label,

(g) detecting the presence or absence of said detectable label of the detectable oligonucleotide extension products, and

10. The method of claim 9, wherein said genomic DNA fragment is unamplified.

1 1 . The method of claim 9, wherein the 5' end of said set of oligonucleotides is affixed to a solid phase matrix.

12. The method of claim 1 1 , wherein said set of oligonucleotides affixed to the solid phase matrix further comprises a component selected from the group consisting of a nucleotide derivative, and a spacer.

13. The method of claim 9, wherein said polymerization agent is a 3'-5' exonuclease minus DNA polymerase.

14. The method of claim 9, wherein said detectable label is selected from the group consisting of biotin, fluorescence, and radioisotope.

15. A kit for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid comprising, in a packaged combination,

(a) an oligonucleotide or a set of oligonucleotides having a sequence of interest, wherein said oligonucleotide(s) is(are) selected so as to be sufficiently complementary to a strand of a sample nucleic acid to anneal therewith,

(b) a polymerization agent, and

(c) nucleotides having a detectable label.

16. The kit of claim 15, wherein said specific nucleotide is a SNP.

17. The kit of claim 15, further comprising a reaction buffer.

18. The kit of claim 15, further comprising a solid phase matrix.

19. An oligonucleotide for determining the identity of a specific nucleotide at a defined site of interest in a sample nucleic acid having an allele specific nucleotide at the 3' end, the 5' end affixed to a solid phase matrix and a sequence complementary to a sequence in a sample nucleic acid.

20. The oligonucleotide of claim 17, wherein said solid phase matrix is a silicon based chip.