WO2003018835A2

WO2003018835A2 - Method for rapid detection of haplotypes

Info

Publication number: WO2003018835A2
Application number: PCT/DK2002/000552
Authority: WO
Inventors: Mogens Fenger; Joan Bentzen
Original assignee: Hvidovre Hospital
Priority date: 2001-08-23
Filing date: 2002-08-22
Publication date: 2003-03-06
Also published as: WO2003018835A3

Abstract

The present invention describes a method and kits for determining the genetic haplotype (linkage phase) of a part of an individual genotype, by determining the presence or absence of two or more specific nucleotide polymorphisms on one of a pair of homologous chromosomes.

Description

Method for rapid detection of hapiotypes

Field of invention

The present invention relates to the field of genetic analysis and diagnostics, in particular to enhanced methods of determining the haplotype of an individual. This application claims priority from Danish Patent Application number PA 2001 01252 filed 23 August 2001 , which is hereby incorporated by reference in its entirety. All patent and non-patent references cited in that application, or in the present application, are also hereby incorporated by reference in their entirety.

General background

With the advent of the modern molecular genetics, it is becoming increasingly clear that a substantial part of human diseases have a genetic component. Whereas the initial discoveries within the field of medical genetics were dominated by relatively rare diseases characterised by a strong one-mutation one-disease relation, emphasis is now being put on the investigation of frequent diseases such as diabetes, dyslipidemia, hypertension and cardiovascular diseases, wherein the one-mutation: one-disease relation does not exist in the majority of cases. Most of these common diseases are polygenic, involving several loci, and many population association studies leave little doubt that the genotype of a number of the involved loci must be established in order to provide reliable data for use in diagnosis, prognosis and pharmacogenetics.

The haplotype is the set of alleles borne on one of a pair of homologous chromosomes. Often the particular combination of alleles in a defined region of some chromosome, e.g. the locus of the major histocompatability complex (MHC), is referred to as the haplotype of that locus. The central dogma of modern molecular genetics teaches that it is the haplotype of the coding part of a gene that determines the amino acid sequence and thus the function of the resulting protein. In connection with studies aimed at establishing an association between the risk of developing a particular disease and the genetic makeup of the patients, information of the haplotype-investigations are superior to conventional genetic investigations of only single loci, because the haplotype provides physiological relevant information from more loci. Polymorphisms and mutations are ubiquitous in the genome. The distinction between polymorphisms and mutations lies in the frequency of alleles in a gene, where a mutation is defined if one allele amounts to more than 99% of the alleles, and polymorphisms accounts for all other situations. Both polymorphisms and mutations can be single nucleotide substitutions, deletions, insertions or rearrangements. As a consequence of the Human Genome Project interest has been focussed particularly on single nucleotide polymorphisms (SNP) for various reasons including their usage in genome scanning for diseases-related genes. More than 2 million SNP's has been detected, some but not all of which are directly related to diseases.

Often more than one polymorphism, in particular SNPs, are present in the same gene. The significance of this varies, but in coding regions, i.e. regions of the gene coding for the proteins, this is of particular interest: in diploid organisms as human it is of importance on which of the two alleles the polymorphisms are located, as this will determine of 0, 1 or several changes in the same protein is present and consequently influence the function of the protein differently.

A number of methods exist that can be used to detect single base mutations and polymorphisms. Detailed description of useful methods may be found in Ausubel et al. Current protocols in molecular biology, (2000) John Wiley and Sons, Inc., N.Y. and in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Among the more important methods are: DNA sequencing; single strand conformation polymorphism (SSCP) method; denaturing gradient gel electrophoresis (DGGE) method; dideoxy fingerprinting, restriction endonuclease fingerprinting and constant denaturing gel electrophoresis (CDGE). However, all of these methods are labour-intensive, involves fairly complex handlings, and are not particular suited for routine haplotyping.

Mutations can be detected by specific hybridisation of oligonucleotides to the nucleic acid to be analysed.

US 6,022,686 describes the simultaneous use of more fluorescent probes for the detection of mutations by liquid phase hybridisation. Since the detection occurs by hybridisation of nucleic acid sequences in homogeneous solution, the method will result in detection of probe specific sequences from both chromosomes. Thus, it does not seem to be easily applicable for determination of haplotypes.

Variations in DNA-sequences may also be detected by hybridisation in a system consisting of a solid and a liquid phase (two-phase systems). One particular useful approach is the so-called "sandwich" hybridisation described in US 4,486,539. Many haplotypes are described by single base polymorphisms, but US 4,486,539 do not provide methods to detect single base polymorphisms. This is however possible with the method described in US 5,633,134 wherein mutations are detected by hybridisation in the presence of a quaternary ammonium salt is described. This piece of art also devise methods for detection of multiple mutations in one gene but only by hybridisation in the presence of a quaternary ammonium salt, and the use of the method for detection of haplotypes is not described.

A crucial point in determining the haplotype by molecular analysis is to resolve the arrangement of polymorphisms into definitive haplotypes for each chromosome. None of the above mentioned methods has addressed this issue specifically.

The resolution issue was however addressed by Ruano et al. (Proc Natl Acad Sci USA 87, 6296-6300). In this paper Ruano et al. obtain the resolution of haplotypes by diluting a sample of chromosomal DNA to a degree that ensures that DNA from only one chromosome is amplified in the subsequent polymerase chain reaction (PCR) amplification process. In later publications the same group resolve the haplotype by allele specific PCR, see e.g. Michalatos-Beloin et al., Nucleic Acid Res 24, 4841-4843. In both the two approaches the actual polymorphisms are detected by time-consuming gel analysis not suited for routine haplotyping.

An elegant method for determining the haplotype directly by use of high resolution single- walled carbon nanotube (SWNT) atomic force microscopy (AFM) has recently been published (Woolley et al (2000); Nature Biotechnology; 18; 760-763). In this method labelled oligonucleotides are hybridised specifically to complementary target sequences in template DNA, and the positions of the tagged sequences are detected by direct SWNT tip imaging. The method is suitable even for smaller DNA fragments (> 5 kilo bases), but requires expensive instruments and is time consuming. Several references in the prior art address the problem of determining whether a diploid subject is a homo- or heretozygote for a given mutation. Examples of such references include WO 99/09210, which discloses an example of allele-specific PCR. The amplicons are captured on a solid surface in different places and followed by hybridisation with one common detection probe. From the ratio between the two signals (two alleles) at the two positions it can be determined whether an individual is a homo- or heterozygote.

WO 00/26412 discloses a method for capturing amplified nucleotide sequences with allele-specific capture probes. The captured nucleotide sequences are visualised with a labelled stacking probe, which is hybridised to the target nucleotide sequence in tandem with the capture probe. It is possible using the disclosed method to determine whether an individual is a homo- or heterozygote for a given polymorphism.

WO 00/20644 also discloses the use of allele-specific capture probes, and WO 00/24926 discloses the detection of alleles using allele specific primers only.

WO 00/39331 discloses allele specific primers for amplification of specific alleles and allele specific probes for detection of one or more of three polymorphisms in the PDK2 gene.

None of these references address the problem of determining the linkage phase between two or more polymorphisms, but merely address determination of homo- versus heterozygosity.

Another reference relating to detection of two polymorphisms is WO 92/16180. The reference discloses a method for detecting ATZ resistance in HIV. The method is based on the use of nucleotide probes for detecting two closely linked polymorphisms lying within 10 nucleotides from one another. It is stressed that these polymorphisms must be located symmetrically on the probe. HIV being a virus by nature has only one genome, which is not a chromosome. Therefore the reference does not touch upon the resolution of linkage phase, since two polymorphisms are always linked in a virus.

Thus, the existing methods for determining haplotypes have significant limitations and further improvements are greatly needed for their use in large-scale genetic screening of clinical material. Definitions

Polymorphism: in the context of the present invention the term polymorphism is also intended to cover a mutation.

Set of oligonucleotide probe members: the term probe is used in the meaning that the term normally has in molecular biology. In the context of the present invention the term probe only covers the sequence which hybridises to the target nucleotide sequence. It being understood that other entities can be linked to the probe, e.g. linkers, labels, a member of an affinity pair, non-hybridising nucleotide sequence, etc. It is also to be understood that two members may be linked to each other via e.g. a spacer sequence, so that the set of members are comprised within the same molecule.

Capture probe: an oligonucleotide probe, which is or can be linked to a solid surface.

Detection probe: an oligonucleotide probe, which is labelled.

Summary of the invention

The present application describes a method for fast, inexpensive and uncomplicated determination of haplotypes in nucleic acid fragments of varying length (from 25 to more than 40.000 base pairs).

According to the invention, a sample of nucleic acid is either directly or following an amplification step by polymerase chain reaction (PCR) or any other method that performs amplification of specific nucleic acid fragments contacted with allele specific oligonucleotides under conditions, which allow the detection of single base differences. By the procedures of the invention it is possible to obtain a hybridisation signal from only one of the two corresponding nucleic acid fragments of a chromosome pair, thus obtaining information about the haplotype of said nucleic acid fragments.

In a first aspect the invention relates to a method for detecting the presence or absence of multiple haplotypic genetic variations in a preselected region of one chromosome of a chromosome pair, comprising the steps of: a) selecting a multiplicity of nucleotide polymorphisms suspected to occur in said preselected region

b) designing a set of oligonucleotide probe members, each of which is capable of hybridising specifically with one individually selected nucleotide sequence in said preselected region comprising at least one polymorphism suspected to occur in the preselected region

c) obtaining from an individual a nucleic acid sample which contains a target nucleic acid comprising the preselected region of the one member or a chromosome pair

d) isolating or specifically analysing at least part of the target nucleic acid of the one member of a chromosome pair comprising the preselected region thereby obtaining a resolution of the haplotypes from the individual

e) detecting the presence or absence of said multiplicity of polymorphisms in said preselected region by nucleic acid hybridisation with said set of oligonucleotide probe members.

For the purpose of the present invention, a "set or probe members" in intended to cover two or more probe members.

Through the use of oligonucleotide probe members, which each hybridise specifically to a sequence comprising a polymorphism or mutation a simple and rapid method for the determination of the haplotype of an individual is provided. One of the core steps of the method is the resolution of the haplotype or linkage phase to make sure that what is determined is the presence or absence on only one chromosome of a chromosome pair. The resolution may be carried out in different ways. Common for these is that only standard laboratory equipment and standard molecular biology methods are used, so that the determination can be carried out simply and rapidly.

Different embodiments of the method are provided, which each result in the resolution of haplotypes and in the determination of the presence or absence of a multiplicity of polymorphisms in the preselected region. In a first embodiment, a) one probe of the set is an allele specific capture probe being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific detection-probe being specific for one allele of a second polymorphism in said preselected region, c) isolating at least part of the target nucleic acid sequence comprises capturing the target nucleic acid sequence with the allele specific capture probe, obtaining a resolution of the haplotype, d) detecting the presence or absence of said multiplicity of polymorphisms comprises contacting the amplified target nucleic acid with the allele specific probe, and detecting the hybrid between the target nucleic acid and the allele specific detection probe.

According to this embodiment, target nucleotide sequence carrying a specific allele in the first locus is captured, and target DNA also carrying a specific allele in the second locus is detected.

In a second embodiment, a) one probe of the set is an allele-specific primer being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific detection probe being specific for one allele of a second polymorphism in said preselected region, c) isolating the target nucleic acid comprises amplifying it using a primer pair of which one member is said allele specific primer, obtaining a resolution of the haplotypes, d) detecting the presence or absence of said multiplicity of polymorphisms comprises contacting said target with the allele specific detection probe, and detecting the hybrid between the amplified target nucleic acid and the allele specific detection probe.

According to this embodiment, target nucleotide sequence carrying a specific allele in the first locus is amplified, and target DNA also carrying a specific allele in the second locus is detected. This may be done by capturing the amplified target, by immobilising the amplified target, or in solution.

In a further embodiment a) the set of oligonucleotide probe members are assembled into a multifunctional detection probe, said multifunctional detection probe comprising

• at least a first allele specific nucleotide sequence, said first allele specific nucleotide sequence being specific for one allele of a first polymorphism, • at least a second allele specific nucleotide sequence, said second allele specific nucleotide sequence being specific for one allele of a second polymorphism,

• a spacer sequence, which does not hybridise to any part of the target nucleic acid under conditions where the allele specific sequences hybridise, and which separates said first and second allele specific nucleotide sequences, and • a detectable label, b) specifically analysing the target nucleic acid comprises contacting the target with the multifunctional detection probe, obtaining a resolution of the haplotypes, and c) detecting the absence or presence of said multiplicity of polymorphisms comprises detecting the hybrid between said multifunctional probe and the target nucleic acid.

According to this embodiment, target nucleotide sequence carrying a specific allele in the first locus and a specific allele in the second locus is detected.

In a further embodiment, a) one probe of the set is an allele-specific primer being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific primer being specific for one allele of a second polymorphism in said preselected region, c) isolating the target nucleic acid comprises amplifying it using the primer pair of said allele specific primers, obtaining a resolution of the haplotypes, d) detecting the presence or absence of said multiplicity of polymorphisms comprises contacting said target with a detection probe, and detecting the hybrid between the amplified target nucleic acid the detection probe.

According to this embodiment, only the target nucleotide carrying both specified polymorphisms is amplified. The presence of the target can be determined using a target probe which hybridises to any part of the target, i.e. the detection probe does not have to be allele specific.

In a further embodiment, a) one probe of the set is an allele specific detection probe being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific detection probe being specific for one allele of a second polymorphism in said preselected region, said other probe being differently labelled, c) specifically analysing the target nucleic acid comprises contacting the target nucleic acid sequence with the probes, obtaining a resolution of the haplotypes, d) detecting the presence or absence of said multiplicity of polymorphisms comprises detecting the hybrid between the amplified target nucleic acid and the allele specific detection probes.

According to this embodiment the target nucleic acid is contacted with a multiplicity of differently labelled allele specific detection probes. The presence of two polymorphisms on a target nucleic acid sequence is detected by detecting the labels from all the differently labelled allele specific detection probes. An example of this method is disclosed in example 3 and 4.

The above embodiments may be combined in many ways for the resolution of the linkage phase between more than two polymorphisms. For each polymorphism to be resolved, one probe member for each polymorphism is needed. For 3 polymorphisms, one probe may be an allele specific primer, one may be an allele specific capture probe and one may be an allele specific detection probe. Alternatively, two probes may be allele specific primers, or two probes may be differently labelled detection probes. The use of three differently labelled allele specific probes is also possible.

In a further aspect the invention relates to a kit for detecting the presence or absence of multiple haplotypic genetic variations in a preselected region of one chromosome of a chromosome pair, comprising

a) at least two oligonucleotide probes, each of which is capable of hybridising specifically with one allele of an individually selected polymorphic locus occurring in said preselected region, one of said probes being a detection probe, which is bound to a label, two adjacent polymorphic loci being separated by at least 15 nucleotides, b) buffers and solutions for carrying out hybridisation under conditions of high stringency between the oligonucleotide probes and a target nucleic acid sequence comprising said preselected region.

The prior art does not teach kits for determination of the haplotype or linkage phase of adjacent polymorphisms, which are separated by more than 15 nucleotides. The kit may be used in the method according to the present invention. The kit may either contain a combination of probes to resolve a particular haplotype in order to answer the question: Does the individual have this haplotype yes or no? Alternatively the kit may comprise probes to cover all the possible combinations of the multiplicity of polymorphisms in order to answer the question: Which haplotype does the individual have?

A kit for detecting the presence or absence of multiple haplotypic genetic variations in a preselected region of one chromosome of a chromosome pair, comprising

a) a multifunctional oligonucleotide probe comprising

• a first allele specific nucleotide sequence, said first allele specific nucleotide sequence being specific for one allele of a first polymorphic locus,

• a second allele specific nucleotide sequence, said second allele specific nucleotide sequence being specific for one allele of a second polymorphic locus,

• a spacer sequence, which does not hybridise to any part of the target nucleic acid under conditions where the allele specific sequences hybridise, and which separates said first and second allele specific nucleotide sequences, and

• a detectable label, b) buffers and solutions for carrying out hybridisation under conditions of high stringency between the oligonucleotide probes and a target nucleic acid sequence comprising said preselected region.

The multifunctional oligonucleotide probes make use of the fact that intramolecular hybridisation is thermodynamically favourable compared to hybridisation between separate molecules. This difference between intra- and inter-molecular hybridisation is in particular significant when hybridisation is performed at low concentrations of nucleic acid and results in a significant difference even between the hybridisation of the bifunctional probe to one or two amplified fragments, the hybridisation to one fragment being the most favourable. Figure legend

Figure 1 : Region spanning the codon 2488 and codon 2712 loci in the apolipoprotein B gene (exon 26). The nucleotide sequence of exons 22 to 29 (Genbank accession No. M19828). The complete sequence of the ApoB gene can be found in Genbank, accession No. NM_000384. Forward primer (SEQ ID No 5) and reverse primer (SEQ ID No 4) define the boundaries of the PCR product confining the preselected region. The Biotin labelled 2488 hybridisation oligos (SEQ ID No 6 and 7) are immobilised onto a solid surface and subsequently hybridised under stringent conditions to the preselected region. The labelled 2712 hybridisation oligos (SEQ ID No 2 and 18) are hybridised to the DNA in an analogous procedure. Unhybridised oligos are removed by washing and the oligos that remain attached are detected by their emission of fluorescent light.

Figure 2:

Region spanning the codon 2488 and codon 2712 loci in the apolipoprotein B gene. The two forward allele specific primers (SEQ ID No 16 and 17) and the reverse primer (SEQ ID No 4) define the boundaries of the PCR product confining the preselected region. Cy3.5 or Acridin Orange labelled 2712 hybridisation oligo (SEQ ID No 2 and 18) hybridise to the region containing the codon 2712 polymorphism.

The biotin labelled 2488 primers are built into the PCR product and subsequently used to immobilise the fragment to a solid surface. Following the immobilisation the DNA is hybridised with Cy3.5 labelled 2712 hybridisation oligos. Unhybridised oligos are removed by washing and the oligos that remain attached are detected by their emission of fluorescent light.

Figures 3A and 3B

The long waved line illustrates the amplified DNA. The two arrows point to the sites of the polymorphisms. The figure illustrates that the labelled probe is stably hybridised to the target DNA if and only if there is a 100% match to the two polymorphisms simultaneously (3A). If the probe does not hybridise to one or both of the two polymorphisms no stable complex will be formed. Non-hybridised labelled probes are removed and hybridised probes are detected (see text for details). The star illustrates that the probe is labelled.

Figure 3C

Four probes are synthesised including all combinations of SNPs (bold letters). Any combination of haplotype detection can be performed in one to four detection tubes using the appropriate amount of different labels i.e. if the detection is performed for all combinations in one tube four different labels are needed, while the labels can be the same if four detection tubes are used.

The two SNPs are flanked by 3 complementary nucleotides on each site of the SNPs. The spacer does not have any or low homology to the intervening sequence between the two SNPs in the target DNA.

Figure 4

The long waved line illustrates the amplified DNA. The two arrows point to the site of the polymorphisms. The target DNA is amplified using allele specific primers covering the polymorphism (in this case the first polymorphic area). A primer is used for each allele in separate amplification reactions. The second polymorphism is detected by allele specific labelled probes.

Detailed disclosure of the invention.

The present invention relates to a method of detecting a multiplicity of specific polymorphisms that occur on the same physical chromosome thus providing detailed information of the haplotype-phase of a specific region of the chromosome that has been selected on beforehand. The term "preselected region" describes this region. The size of the preselected region may vary. According to the invention the preselected region may vary between 25 bp and up to 2000 kbp, such as 25-1500 kbp, or 25-1000kbp, 25-500 kbp, 25-250 kbp, 25-100 kbp, 25-75.000 bp, 25-50.000 bp, 25-40.000 bp, 25-40.000 bp, 25-40,000 or 25-10,000, 25-5,000, 25-1000, 25-500, 25-300, 25-200 or 25-100 nucleotides in length. The term "a multiplicity" is herein defined as two or more. The core of the invention is the procedure for resolution of haplotypes or linkage phase from a sample of individual genomic DNA by detecting the presence of absence of a multiplicity of known polymorphisms only on one chromosome of a chromosome pair.

Here "polymorphism" refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population

One particular interesting type of polymorphisms is the so-called single nucleotide polymorphisms (SNPs). "SNPs" are defined as polymorphisms in which the sequence between two alleles only differs by one single nucleotide. Presently, e.g. in relation to the Human Genome Project, large numbers of SNPs are mapped and gathered in various catalogues, some of these being freely accessible and form a data source of immense value.

In a preferred embodiment of the invention the distance between two adjacent polymorphisms is at least 15 nucleotides. The reason for this being that when polymorphisms are located very close to each other (e.g. a few up to 10 nucleotides) then the linkage between the polymorphisms is so strong that presence of one allele in a particular position often means that the allele of the closely positioned locus can be predicted without further analysis. The longer the distance between loci, the weaker is the linkage between them and the higher is the need to be able to determine the linkage phase.

The methods of the present invention can also be used for determining the linkage phase between two adjacent loci, when two adjacent polymorphisms are separated by at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 150 nucleotides, such as at least 200 nucleotides, for example at least 250 nucleotides, such as at least 300 nucleotides, for example at least 400 nucleotides, such as at least 500 nucleotides, for example at least 750 nucleotides, such as at least 1000 nucleotides, for example at least 2000 nucleotides, such as at least 5000 nucleotides, for example at least 10000 nucleotides, such as at least 20000 nucleotides, for example at least 30 kb, such as at least 40 kb, for example at least 50 kb, such as at least 75 kb, for example at least 100 kb, such as at least 250 kb, for example at least 500 kb, such as at least 750 kb, for example at least 1000 kb. The upper limit is determined by the maximum length of a DNA strand that can be isolated or amplified.

The probes used in the present invention may consist of any kind of nucleotides, although for some purposes the use of one or more LNA monomers is preferred. The stretch of nucleotides which is complementary to a sequence in the target nucleotide should be long enough to ensure that hybridisation is specific, i.e. that the probability of a similar sequence in another position of the target nucleotide is approximately 0. According to a preferred embodiment of the invention, the length of a probe is from 5 to 40 nucleotides, preferably 7 to 20 nucleotides.

Example 1 illustrates one preferred embodiment of the invention in which part of the gene of interest is analysed. The part that is to be studied is defined by the two PCR primers (see figure 1) used for the amplification of a preselected region of the gene. In other embodiments other regions of other genes may be selected. However in the present context it is important to select a region of the genome, herein defined as the "preselected region" and furthermore to select two or more specific polymorphisms that may occur in the selected region. This embodiment is exemplified by a procedure for determining if in a sample of genomic DNA the SNPs at codon 2488 (C->T) and 2712 (T->C) of the human gene for apolipoprotein B-100 occur on the same physical chromosome thus in essence determining what herein is defined as the "ApoB 2488/2712 haplotype".

In a specific example of the first embodiment (example 1) the resolution into haplotypes is performed by hybridising the PCR amplified preselected region of the ApoB gene to a set consisting of two probes (SEQ ID No 6) and (SEQ ID No 7) which are specific for the codon 2488 (C) or the codon 2488 (T) allele of the human ApoB gene, respectively. The probes, which are termed the "capture probes", are in example 1 coupled to a biotin molecule allowing them subsequently to be immobilised to the solid surface of streptavidin coated microtiter plate wells. To obtain the resolution, the two capture probes are immobilised in different wells of the microtiter plate, the amplified preselected region is hybridised to the capture probes under stringent conditions followed by a stringent hybridisation with two probes, one that is specific for the ApoB codon 2712 (T) allele (SEQ ID No 2) and is labelled with acridin orange and one that is specific for the ApoB codon 2712 (C) allele (SEQ ID No 18) and labelled with Cy3.5. The two different labels, acridin orange and Cy3.5, have fairly different emission spectres allowing the 2488 (C/T), 2712 (C/T) haplotype of a sample to be established from the fluorescence signal from two corresponding wells as described in example 1.

The sample which contains the nucleic acid to be studied, herein defined as the "target nucleic acid" may be genomic DNA, including chromosomal DNA and mitochondrial DNA as well as mRNA and other types of RNA which can be copied into cDNA by a reverse transcription reaction. Although the main focus of the present invention are haplotype determinations of mammals, it is further contemplated that any types of diploid organisms e.g. non-mammalian animals, plants, algae, yeasts and fungi or mixtures of haploid organisms can be haplotyped by the methods described herein. In the case of chloroplast- containing organisms chloroplast DNA may be an interesting source of target nucleic acid as is also mitochondrial DNA.

In the present context, the term "label" means a group which is coupled to the nucleic acid and which can be used for the detection or other subsequent reactions e.g. immobilisation of the nucleic acid. The oligonucleotides may be labelled by a number of methods well known in the art. Conveniently, oligonucleotides may be labelled during their solid-phase synthesis using any of the many commercially available phosphoramidite reagents for 5' labelling. Illustrative examples of oligonucleotide labelling procedures may be found in US Pat 6,255,476.

A wide variety of appropriate indicators are known in the art, including fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, which are capable of giving a detectable signal. A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure.

A particular fluorescent label has a characteristic excitation and emission spectre which allows the simultaneous detection of several different fluorescent labelled molecules if the labels are selected appropriately.

A large number of different useful fluorescent labels are given in the art and may be selected from the group comprising, but not limited to: Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7

(trademarks for Biological Detection Systems, Inc.), fluorescein, acridin, acridin orange, Hoechst 33258, Rhodamine, furthermore: Rhodamine Green, Tetramethylrhodamine, Texas Red, Cascade Blue, Oregon Green, Alexa Fluor (trademarks for Molecular Probes, Inc.), 7-nitrobenzo-2-oxa-1 -diazole (NBD), pyrene and Europium, Ruthenium, Samarium, and other rare earth metals).

Other potential labelling moieties include, radioisotopes, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers, magnetic labels, and linked enzymes.

In preferred embodiments, one will likely desire to employ a fluorescent label or an enzyme tag, such as urease, alkaline phosphatase or peroxidase, instead of radioactive or other environmental undesirable reagents. In the case of enzyme tags, colorimetric indicator substrates are known that can be employed to provide a means visible to the human eye or spectrophotometrically, to identify specific hybridisation with complementary nucleic acid-containing samples.

The most preferred embodiment of the invention is illustrated by example 2. Both example 1 and example 2 comprises binding of the nucleic acid to a solid surface. In both examples the immobilisation is obtained by coupling a biotin molecule to the nucleic acid and subsequently immobilising the nucleic acid on a streptavidin modified surface. However, the nature of the means for immobilisation and of the support is a matter of choice. Numerous suitable supports and methods of attaching nucleotides to them are well known in the art and widely described in the literature. Thus for example, supports in the form of microtiter wells, tubes, dipsticks, particles, fibres or capillaries may be used, made for example from agarose, cellulose, alginate, teflon, latex or polystyrene. Conveniently, the support may comprise magnetic particles, which permits the ready separation of immobilised material by magnetic aggregation.

The solid support may carry functional groups such as hydroxyl, carboxyl, aldehyde or amino groups for the attachment of nucleotides. These may in general be provided by treating the support to provide a surface coating of a polymer carrying one of such functional groups, e.g. polyurethane together with a polyglycol to provide hydroxyl groups, or a cellulose derivative to provide hydroxyl groups, a polymer or copolymer of acrylic acid or methacrylic acid to provide carboxyl groups or an amino alkylated polymer to provide amino groups. US 4,654,267 describes the introduction of many such surface coatings. Alternatively, the support may carry one member of an "affinity pair", such as avidin, while the amplified DNA is conjugated to the other member of the affinity pair in casu biotin. Representative specific binding affinity pairs are shown in Table 1.

The streptavidin/biotin binding system is very commonly used in molecular biology, due to the relative ease with which biotin can be incorporated within nucleotide sequences, and indeed the commercial availability of biotin-labelled nucleotides, and thus biotin represents a preferred means for immobilisation.

In a preferred embodiment of the invention, one of the amplified DNA strands is labelled with a molecule, which is subsequently used to immobilise the labelled DNA strand to a solid surface as described in Example 2 herein.

The DNA may be labelled by a number of methods. One convenient method to label DNA is to enclose one labelled amplification primer oligonucleotide in the amplification reaction mixture. During the amplification process the labelled oligonucleotide is built into the DNA fragment which becomes labelled.

During synthesis, oligonucleotides may also be labelled or coupled to chemoreactive groups comprising, but not limited to: sulfhyl, primary amine or phosphate. The subsequent use of such labelled primers in PCR, LCR or similar oligonucleotide dependent amplification methods results in labelled DNA fragments which can be immobilised on specialised surfaces. For instance SH-modified DNA may be immobilised on a gold surface (Steel et al. (2000) Biophys J 79:975-81) likewise 5'-phosphorylated DNA or 5'-aminated DNA may be immobilised by reaction with activated surfaces (Oroskar et al. (1996) Clin Chem 42:1547-55; Sjoroos et al (2001) Clin Chem 47:498- 504).

During synthesis, oligonucleotides may also be labelled or coupled to photoreactive groups. Acetophenone, benzophenone, anthraquinone, anthrone or anthrone-like modified DNA can for instance be activated by exposure to UV light and immobilised on a wide range of surfaces as described in European and US patents: EP 0820483, US 6,033,784 and US 5,858,653. Also photoreactive psoralens, coumarins, benzofurans and indols have been used for immobilisation of nucleic acids. An extensive discussion of immobilisation of nucleic acids can be found in WO 85/04674.

The DNA may be amplified by one of many methods. One of the best known and widely used amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in US 4,683,195, US 4,683,202 and US 4,800,159, however other methods such as LCR (Ligase Chain Reaction, see Genomics (1989) 4:560-569), NASBA (Nucleic Acid Sequence-Based Amplification, see PCR Methods Appl (1995) 4, S177- S184), strand displacement amplification (Current Opinion in Biotechnology (2001 ) 12:21- 27), rolling circle amplification (Current Opinion in Biotechnology (2001) 12:21-27), or non PCR methods such as T7 polymerase amplification can be applied.

If the selected polymorphisms are situated a long distance from each other fairly long fragments of genomic DNA have to be amplified. Several techniques that result in amplification of fairly long fragments of DNA are described in the art. One particular useful procedure is the so-called "long range PCR", which allows amplification of very large fragments (Proc. Natl. Acad. Sci. USA. (1994) 91 : 2216-20; Methods-Mol-Biol. (1997) 67: 17-29.). Kits allowing the amplification of templates up to 40.000 base pair long are commercially available, e.g. the TripleMaster™ PCR System (cat. no. 0032 008.208) of Eppendorf AG, Hamburg, Germany.

In order to obtain phase-specific detection of polymorphisms in the most preferred embodiment illustrated by example 2, the initial amplification process of this embodiment is an allele specific amplification. An "allele specific amplification" is defined as an amplification process resulting in the amplification of one allele only. In the present context the allele specific amplification results in the amplification of a specific part of only one of the chromosomes forming a chromosome pair. Allele-specific amplification can be accomplished in a number of ways. Allele-specific PCR as described in EP 0332435 is a widely used method. Furthermore, a very efficient method is described in WO 00/56916. The use of LCR for allele-specific amplification is described in WO 89/09835 and also the methods of Nucleic Acid Sequence-Based Amplification (NASBA), strand displacement amplification and rolling circle amplification may in principle be modified to perform an allele specific amplification and thus be used to obtain resolution of the haplotypes.

To detect the haplotype resolved DNA, oligonucleotides, complementary to the remaining loci of interest on the DNA fragment, are made as described. A fluorescent label is preferred because it provides a very strong signal with low background. It is also optically detectable at high resolution and sensitivity through a quick scanning procedure. However other labels may be contemplated.

In further embodiments the oligonucleotides are kept in solution and are brought into contact with the DNA targeted for haplotype determination. This may be done as in example 2 but using the allele specific primers SEQ ID No 1 and 3 (codon 2488), which are not biotin-labelled. The oligonucleotides are then allowed to hybridise to the DNA under highly stringent conditions. In the scope of the present invention the term

"hybridisation" signifies hybridisation under conventional hybridising conditions, preferably under stringent conditions, as described for example in Sambrook et al., Molecular

Cloning, A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press,

Cold Spring Harbor, N.Y.). The term "stringent" when used in conjunction with hybridisation conditions is as defined in the art, i.e. 15-20°C under the melting point T_m, cf.

Sambrook et al, 1989, pages 11.45-11.49. Preferably, the conditions are "highly stringent", i.e. 5-10°C under the melting point T_m. Under highly stringent conditions hybridisation only occurs if the identity between the oligonucleotide sequence and the locus of interest is 100 %, while no hybridisation occurs if there is just one mismatch between oligonucleotide and DNA locus. Such optimised hybridisation results are reached by adjusting the temperature and/or the ionic strength of the hybridisation buffer as described in the art. However equally high specificity may be obtained using high-affinity DNA analogues. One such high-affinity DNA analogues has been termed "locked nucleic acid" (LNA). LNA is a novel class of bicyclic nucleic acid analogues in which the furanose ring conformation is restricted in by a methylene linker that connects the 2'-0 position to the 4'-C position. Common to all of these LNA variants is an affinity toward complementary nucleic acids, which is by far the highest reported for a DNA analogue (0rum et a). (1999) Clinical Chemistry 45, 1898-1905; WO 99/14226 EXIQON). LNA probes are commercially available from Proligo LLC, Boulder, Colorado, USA. Another high-affinity DNA analogue is the so-called protein nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone (Science (1991) 254: 1497-1500).

Finally, the haplotype in this preferred embodiment is determined by detection of fluorescence emitted from the different oligonucleotides hybridised to the amplified DNA. This may be accomplished by use of any a number of different fluorescence readers with the appropriate filters. The use of one such reader is described in examples 1 and 2.

In further embodiments of the invention both the resolution of the haplotypes and the detection of the multiplicity of nucleotide polymorphisms are performed in suspension, defined here as a "one-phase system".

In one embodiment this is accomplished by the amplification of the preselected region. The amplified fragment is hybridised to specially designed probes, which are capable of detecting a multiplicity of polymorphisms. Examples of such probes designed to resolve and detect the ApoB 2488/2712 haplotype are given in Figure 3c and Table 2 (SEQ ID No. 10-13). Such "bifunctional" or "multifunctional" probes contain at least 2 stretches of relatively short sequences which are complementary to each of the two studied polymorphisms separated by a region of spacer DNA which will not hybridise with the amplified region under the conditions in the experiment. The length of the spacer sequence is preferably from 8 to 28 nucleotides.

The multifunctional probes may of course span several loci of polymorphism, such as to cover at least 3 polymorphisms, for example at least 4 polymorphisms, such as at least 5 polymorphisms, such as at least 10 polymorphisms. In each case, the sequences hybridising with the target nucleotide sequence are separated by a spacer sequence, which does not hybridise to the target.

To obtain hybridisation signal from only one chromosome of a chromosome pair, the procedure takes advantage of the fact that intramolecular hybridisation is thermodynamically favourable compared to hybridisation between separate molecules. This difference between intra- and inter-molecular hybridisation is in particular significant when hybridisation is performed at low concentrations of nucleic acid and results in a significant difference even between the hybridisation of the bifunctional probe to one or two amplified fragments, the hybridisation to one fragment being the most favourable.

The use of probes designed for the detection of the ApoB 2488/2712 haplotype is illustrated in figure 3a-3b. The probes are illustrated in figure 3c. Under the stringent conditions employed in the hybridisation only probes, which are completely complementary to the sequences, comprising both studied polymorphisms will form stable hybrids.

Following the stringent hybridisation, the oligonucleotides which have not hybridised to a DNA locus are removed from the amplified DNA (e.g. by precipitation, size fractionation, electrophoreses or by centrifugation). Then the haplotype is determined by detection of fluorescence from the different oligonucleotides hybridised to the amplified DNA.

As in the case of the previous embodiments other types of fluorescent labels may be applied.

In a further embodiment of the "one-phase system" the detection of the polymorphisms is performed using probes directed against only one polymorphism while the resolution of the haplotypes is performed by allele specific amplification both procedures being performed in suspension. Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.

Examples

Example 1. Detection of ApoB haplotypes by hybridisation to an immobilised detection probe.

The first embodiment of the invention is illustrated by detection of the two polymorphisms at codon 2488 (C->T) and 2712 (T->C) in the gene for apolipoprotein B-100 (see figure 1 for sequence).

Apolipoprotein B (ApoB) is the non-exchangeable protein of the low-density lipoprotein (LDL) complex that is recognised by the LDL-receptor. In the gene for apolipoprotein B- 100 two polymorphisms exist approximately 670 bp apart in codon 2488 (C->T) and 2712 (T->C) (see figure 1 ).

Purification of DNA Human genomic DNA is isolated from 5 mL full blood, using the DNA Isolation Kit for Mammalian Blood (Roche Molecular Systems cat. no. 1 667 327. Roche Molecular Biochemicals, Hvidovre, Denmark) and closely following the recommendations of the manufacturer.

Amplification

Two primers (See "forward primer" ,(SEQ ID NO. 5 ) and "reverse primer" (SEQ ID NO. 4) in Figure 1) spanning the region of the 2488 and 2712 polymorphisms are designed and PCR amplification is carried out in 200 μ PCR tubes (cat.no. AB-0337, ABgene, Merck Eurolab, Denmark) containing 0.6 μM of each primer (see figure 1 , DNA-technology, Arhus, Denmark), 0.2 mM of each dNTP (Quiagen, Merck Eurolab, Denmark), 1.5 mM MgCI₂ (Applied Biosystems, Denmark). 3 U of AmpliTaq Gold DNA-polymerase and 5 μL of 10XGold Buffer (both Applied Biosystems, Denmark), 200 ng of genomic DNA in TE buffer from persons already genotyped at the two loci. Sterile, filtered water taken from a MilliQ synthesis A-10 system (from Millipore, Glostrup, Denmark) was added to a final volume of 50 μL. The tubes were placed in a PTC-200 termocycler (MJ Research, Merck Eurolab, Denmark) and the following program was run using heated lid: 1 X 92°C for 10 min, 40 X (94°C for 45 sec + 55°C for 45 sec + 72°C for 45 sec), 1 X 72°C for 10 min.

Hybridisation

Two oligonucleotides, 21 base pairs long, biotinylated in the 5^' end which are complementary to either the C-allele or the T-allele of the codon 2488 polymorphisms designed (see figure 2 "2488 hybridisation oligo" for sequence, C-allele (SEQ ID NO. 6), T-allele (SEQ ID NO. 7)). These oligos are attached to a microtiter plate well coated with streptavidin (Perkin Elmer, Denmark, part no. C122-105).

The two oligos are placed on separate surfaces (e.g. the one complementary to the C- allele in one microtiter plate well and the one complementary to the T-allele in a different well).

The PCR amplicon is added to the oligonucleotides attached to the solid surface and allowed to hybridise by adding 20 μL of PCR amplicon in 150 μL of PBST buffer to each microtiter plate well. The PBST buffer is made from 1 tablet Phosphate Buffered Saline (code BR14a, Oxoid Ltd, Basingstoke, England) dissolved in 100 mL sterile, filtered water taken from a MilliQ synthesis A-10 system (Millipore, Glostrup, Denmark) and added 50 μL Tween. The surfaces are shaken at 37°C for 30 minutes and then washed with 200 μL 1 x SSCT buffer 5 times. The 1 X SSCT is made by dilution of a 10X SSCT stock containing 3M NaCI and 0.3 M sodium citrate and 0,1% Tween 20.

The PCR-amplicons, which do not contain a perfect match for the attached oligonucleotide will not have hybridised and is therefore washed off, while the PCR- amplicons, which contain a sequence 100% complementary to the one possessed by the oligonucleotide will have hybridised.

Two different oligonucleotides 20 base pairs long, which are complementary to either the T-allele of the codon 2712 polymorphism or the C-allele of the same polymorphism are designed (see figure 1 "labelled 2712 hybridisation oligo" for sequence, SEQ ID No 2 (T- allele) and 18 (C-allele)). The oligonucleotide complementary to the T-allele is labelled with acridin orange (emission max 525 nm) while the oligonucleotide complementary to the C-allele is labelled with Cy3.5 (emission max 596 nm) (labelled oligonucleotides DNA- technology, Arhus, Denmark). The oligonucleotides are brought into contact with the hybridised PCR product and allowed to hybridise to the DNA strand by adding 0.5 μM of the oligonucleotide in 200 μL PBS buffer containing 0.05% Tween to each surface. The surface is shaken at 37°C for 30 minutes and then washed 5 times with 200 μL of SSCT buffer. The surface is then placed in a flourescens reader (Victor², Perkin Elmer, Denmark). Readings are taken using emission filter 535 nm and 615 nm. A positive reading at 535 nm for the surface on which the oligonucleotide complementary to the C- allele in the codon 2488 polymorphism is attached implies that this amplicon has the haplotype C (in codon 2488) T (in codon 2712). A positive reading at 535 nm for the surface on which the oligonucleotide complementary to the T-allele in the codon 2488 polymorphism is attached implies that this amplicon has the haplotype T (in codon 2488) T (in codon 2712). A positive reading at 615 nm for the surface on which the oligonucleotide complementary to the C-allele in the codon 2488 polymorphism is attached implies that this amplicon has the haplotype C (in codon 2488) C (in codon 2712), while a positive reading at 615 nm for the surface on which the oligonucleotide complementary to the T-allele in codon 2488 is attached implies that the amplicon has the haplotype T (in codon 2488) C (in codon 2712).

Example 2. Detection of ApoB haplotypes by hybridisation to a biotinylated PCR- fragment.

To establish a proof of concept for the second embodiment of the invention the following experiment is designed:

Purification of DNA

Human genomic DNA is isolated as described in examplel .

Amplification Two biotin-labelled primers, specific for the 2488C and 2488T alleles respectively (" allele specific forward primer" in Figure 2 and SEQ ID Nos. 16 and 17 and an unmarked primer ("Unmarked reverse primer" in Figure 2 and SEQ ID No. 4) spanning this region is designed (primers from DNA-technology, Arhus, Denmark) and two PCR amplifications are carried out in 200 μL PCR tubes (cat.no. AB-0337, ABgene, Merck Eurolab, Denmark) - one containing 0.6 μM of the forward primer specific for the 2488C allele and 0.6 μM of the reverse primer and one containing 0.6 μM of the forward primer specific for the 2488T allele and 0.6 μM of the reverse primer (see figure 2, DNA-technology, Arhus, Denmark). Both reactions contain 0.2 mM of each dNTP (Quiagen, Merck Eurolab, Denmark), 1.5 mM MgCI₂ (Applied Biosystems, Denmark). 3 U of AmpliTaq Gold DNA-polymerase and 5 μL of 10XGold Buffer (both Applied Biosystems, Denmark), 200 ng of genomic DNA in TE buffer from persons already genotyped at the two loci. Sterile, filtered water taken from a MilliQ synthesis A-10 system (Millipore, Glostrup, Denmark) is added to a final volume of 50 μL.

The tubes are then placed in a PTC-200 termocycler (MJ Research, Merck Eurolab, Denmark) and the following program is run using heated lid: 1 X 92°C for 10 min, 40 X (94°C for 45 sec + 55°C for 45 sec + 72°C for 45 sec), 1 X 72°C for 10 min.

During the PCR amplification a PCR-fragment that 1) is biotinylated is formed characterised by being biotinylated only on one of the strands of the DNA molecule and 2) contains a sequence specific for one of the two alleles complementary to the allele specific oligos.

Binding biotinylated DNA to immobilised streptavidin

Following the PCR amplification the amplicon-containing reaction mix is dispensed into a PCR tube coated with streptavidin (cat.no 1741772, Roche, Denmark) and the biotin on the one DNA strand is allowed to associate with the streptavidin immobilised in the PCR tube. One hybridisation is performed for each allele specific amplification. An aliquot of 20 μL of PCR amplicon is mixed with 150 μL of PBS buffer with 0.05% Tween 20, the mixture is heated to 100°C for 60 seconds, cooled on ice and dispensed into each streptavidin coated PCR tube. Then the tubes are incubated at 37°C for 30 minutes, washed 5 times with 200 μL 1 X SSCT (see example 1) and hybridised with the detection probes as follows.

Hybridisation

Two oligonucleotides complementary to a 20 base pair sequence spanning the codon 2712 polymorphism are designed (see figure 2, SEQ ID Nos. 2 and 18. The oligonucleotide complementary to the codon 2712C allele (and SEQ ID No. 18) is labelled with Cy3.5 (emission max 596 nm) while the oligonucleotide complementary to the codon 2712 T-allele (SEQ ID No. 2) is labelled with acridin orange (emission max 525 nm) (both oligos are from DNA-technology, Arhus, Denmark).

The oligonucleotides are brought into contact with the immobilised PCR product and allowed to hybridise to the DNA. Briefly, 0.5 μM of each hybridisation oligo in 200 μL of PBST buffer (made from 1 tablet Phosphate Buffered Saline (code BR14a, Oxoid Ltd, Basingstoke, England) dissolved in 100 mL sterile, filtered water taken from a MilliQ synthesis A-10 system (Millipore, Glostrup, Denmark) and added 50 μL Tween) is added to each tube. The tubes are shaken at 37°C for 30 minutes and then washed with 200 μL SSCT buffer 5 times.

Detection

The PCR tube is placed in a fluorescence reader (Victor², Perkin Elmer, Denmark) and readings are taken using emission filters 615 nm (for Cy3.5) and 535 nm (for Acridin orange). Haplotype C (in codon 2488) T (in codon 2712) will result in a fluorescence signal from the 535 nm filter in the tube where the 2488C allele specific PCR-amplicon is hybridised, haplotype C (in codon 2488) C (in codon 2712) will result in detection of fluorescent light of 615 nm in the tube where the 2488C allele specific PCR-amplicon is hybridised, haplotype T (in codon 2488) T (in codon 2712) will result in emission of fluorescent light at 535 nm in the tube where the 2488T allele specific PCR-amplicon is hybridised, while haplotype T (in codon 2488) C (in codon 2712) will give a reading from the 596 nm filter in the tube where the 2488T allele specific PCR-amplicon is hybridised.

Example 3. Detection of Apolipoprotein E (apoE) haplotypes by hybridisation to a biotinylated PCR-fragment.

Apolipoprotein E is a constituent of chylomicrons, VLDL, IDL and HDL, which is of central importance to the metabolism of lipiproteins and in particular has a role in receptor recognition. The complete coding sequence of ApoE is disclosed in Genbank, accession number K 00396.

To establish a proof of concept for the invention using two flourophore-labeled probes the following experiment is designed: Purification of DNA

Human genomic DNA is isolated as described in examplel .

Amplification Two primers, specific for the the apolipoproetin E gene (SEQ ID No. 19 and 20) spanning the region in the exon 4 of the apoE-gene including codon 112 and 158 are designed (primers purchased from DNA-technology, Arhus, Denmark). SEQ ID No 19 is biotinylated in the 5'-end.

Three PCR amplifications are carried out in 100 μ\ PCR tubes (ABgene, purchased from Merck Eurolab, Denmark) containing 200 ng of genomic DNA in TE buffer , 0.5 μM of each primer, 0.2 mM of each dNTP, 1.5 mM MgCI₂, 1 U Taq-polymerase and 2,5 μL Goldbuffer, all purchased from Applied Biosystems, Denmark. Water was added to a final volume of 25 μl.

Then the tubes are placed in a PTC-200 termocycler (MJ Research, purchased from Merck Eurolab, Denmark) and the following program is run using heated lid: 1 X 94° C for 10 min, 35 X (94° C for 40 sec + 63° C for 40 sec + 72° C for 40 sec), 1 X 72° C for 10 min.

During the PCR amplification a PCR-fragment that 1) is biotinylated is formed characterized by being biotinylated only on one of the strands of the DNA molecule and 2) contains the sequence including the two polymorphisms defining the alleles of apolipoprotein E.

Binding biotinylated DNA to immobilized streptavidin

Following the PCR amplification the amplicon-containing reaction mixes are dispensed each into a PCR tube coated with streptavidin (cat.no 1741772, purchased from Roche, Denmark) and the biotin on the one DNA strand is allowed to associate with the streptavidin immobilized in the PCR tube. An aliquot of 20 μ\ of PCR amplicon is mixed with 150 μ\ of PBS buffer with 0.05% Tween 20, and the mixture is dispensed into each streptavidin coated PCR tube. Then the tubes are incubated at 37° C for 30 minutes, heated to 100°C for 60 seconds, cooled on ice and washed 5 times with 200 μ\ 1 X SSCT (see example 1 ) and hybridized with the detection probes as follows. Hybridisation

Two oligonucleotides complementary to the codon 112 polymorphism and two oligonucleotides complementary to the codon 158 polymorphism are designed (SEQ ID 21 , 22, 23, 24; Table 2).

The two codon 112 specific detection probes (SEQ ID No 21 and 22) are labeled with Cy3.5 (emission max 596 nm) and the two codon 158 specific detection probes (SEQ ID No 23 and 24) are labeled with acridin orange (emission max 525 nm). The oligonucleotides are brought into contact with the immobilized PCR product and allowed to hybridize to the DNA. Briefly, 0.5 μM of each hybridization oligo in 200 μ\ of PBST buffer (made from 1 tablet Phosphate Buffered Saline (code BR14a, purchased from Oxoid Ltd, Basingstoke, England) dissolved in 100 ml sterile, filtered water taken from a MilliQ synthesis A-10 system (from Millipore, Glostrup, Denmark) and 50 μ\ Tween 20 is added to each tube. The tubes are incubated at 37° C for 5 minutes and then washed with 300 μ\ SSCT buffer 5 times. The detection oligonocleotides are added to the three tubes containing the immobilized target DNA according to the following scheme: SEQ ID No 21 and SEQ ID No 23 are added to the first tube; SEQ ID No 22 and 23 are added to the second tube; and SEQ ID No 22 and 24 are added to the third tube.

Detection

The PCR tube is now placed in a fluorescence reader (Victoi², Perkin Elmer, Denmark) and readings are taken using emission filters 615 nm (for Cy3.5) and 535 nm (for Acridin orange).

Only tubes with fluorescence signals from both the flourophores are diagnostic. That is if double positive signals are obtained in the first tube, the apoE2-allel is present in the sample; if double positive signals are obtained in the second tube, the apoE3-allel is present in the sample; and finally if double positive signals are obtained in the third tube, the apoE4-allel is present in the sample.

Example 4. Detection of Apolipoprotein B (apoB) haplotypes by hybridisation to a biotinylated PCR-fragment. The example was carried out as for example 3 but with the following probes for amplification and hybridisation: Amplification:

Forward primer: SEQ ID No 55' labelled with biotin Reverse primer: SEQ ID No 4 Detection probes:

Codon 2712: SEQ ID No 14 and 15, where the label is Cy3,5. Codon 2488: SEQ ID No 8 and 9, where the label is Acridin Orange.

Table 2: List of oligo sequences

SEQ ID Nol : 5' AGGCCAAATTCCGAGAGACC 3' ApoB codon 2488 C allele primer

SEQ ID No2 : Acridin orange-5 'GGAATTCTAGTATGTGAAGG 3' ApoB codon 2712 C allele detection oligo

SEQ ID No3 : 5' AGGCCAAATTCCGAGAGACT 3' Apo B codon 2488 T allele primer

SEQ ID No4 : 5' TGCTCCGTTCTCAGGTACTTGC 3' ApoB reverse primer

SEQ ID No5 : 5' GATGAAACCAATGACAAAATCC 3' ApoB forward primer

SEQ ID No6: Biotin 5' TATCTTCTAGGGTCTCTCGG 3' ApoB codon 2488 C allele capture probe

SEQ ID No7: Biotin 5' TATCTTCTAGAGTCTCTCGG 3' ApoB codon 2488 T allele capture probe

SEQ ID No8 : Label-5' ttctagAgtctct 3' ApoB codon 2488 T allele detection probe

SEQ ID No9 : Label-5' ttctagGgtctct 3' ApoB codon 2488 C allele detection probe SEQ ID Nol O : Label 1-5 ' tctAgtafcatatafcatafcafcattagGgtc ApoB codon 2712/2488 T/C detection probe

SEQ ID Noll : Label 2-5 ' tctAgtafcafcatafcafcatatafctagAgtc ApoB codon 2712/2488 T/T detection probe

SEQ ID Nol2 : Label 3-5 ' tctGgtatatatatatatafcafctagGgtc ApoB codon 2712/2488 C/C detection probe

SEQ ID Nol3 : Label 4-5 ' tctGgtatafcatatatatatafctagAgtc ApoB codon 2712/2488 C/T detection probe

SEQ ID Nol4 : Label-5' aattctAgtatgt ApoB codon 2712 T allele detection probe

SEQ ID Nol5 : Label-5' aattctGgtatgt ApoB codon 2712 C allele detection probe

SEQ ID Nol 6: Biotin 5' AGGCCAAATTCCGAGAGACC 3' ApoB codon 2488 C allele specific forward primer

SEQ ID Nol 7 : Biotin 5' AGGCCAAATTCCGAGAGACT 3' ApoB codon 2488 T allele specific forward primer

SEQ ID Nol8 : Cγ3.5-5 ' -GGAATTCTGGTATGTGAAGG-3 ' ApoB codon 2712 C allele detection probe

SEQ ID Nol9 Biotin-5 ' -GCGCTGATGGACGAGACCATGAAG ApoE exon4 biotin- labelled forward primer

SEQ ID No20 5 ' CCTGTTCCACCAGGGGC ApoE exon 4 reverse primer

SEQ ID No21 Cy3 . 5-5 ' -G^LC^LC^LG^LC^LA^LC^LA^LC ApoE codon 112 T allele detection probe SEQ ID No22 Cy3.5-5 ' -GC^LC^LG^LC^LG^LC^LAC ApoE codon 112 C allele detection probe

SEQ ID No23 Acridin orange- 5 ' -CA^LGGC^LA^LC^LTT ApoE codon 158 T allele detection probe

SEQ ID No24 Acridin orange- 5 ' -C^LA^LG^LG^LC^LGC^LT^LT ApoE codon 158 C allele detection probe

L = LNA-monomer

Claims

1. A method for detecting the presence or absence of multiple haplotypic genetic variations in a preselected region of one chromosome of a chromosome pair, comprising the steps of:

a) selecting a multiplicity of nucleotide polymorphisms suspected to occur in said preselected region

2. A method according to claim 1 , wherein

a) one probe of the set is an allele specific capture probe being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific detection-probe being specific for one allele of a second polymorphism in said preselected region, c) isolating at least part of the target nucleic acid sequence comprises capturing the target nucleic acid sequence with the allele specific capture probe, obtaining a resolution of the haplotype, d) detecting the presence or absence of said multiplicity of polymorphisms comprises contacting the amplified target nucleic acid with the allele specific probe, and detecting the hybrid between the target nucleic acid and the allele specific detection probe.

3. The method according to claim 2, further comprising providing capture probes specific for every allele of a first polymorphism wherein said capture probes are immobilised in different positions to enable the resolution of both haplotypes of a diploid individual.

4. A method according to claim 1 , wherein

a) one probe of the set is an allele-specific primer being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific detection probe being specific for one allele of a second polymorphism in said preselected region, c) isolating the target nucleic acid comprises amplifying it using a primer pair of which one member is said allele specific primer, obtaining a resolution of the haplotypes, d) detecting the presence or absence of said multiplicity of polymorphisms comprises contacting said target with the allele specific detection probe, and detecting the hybrid between the amplified target nucleic acid and the allele specific detection probe.

5. The method according to claim 4, further comprising amplification of every allele of a first polymorphism in separate amplification reactions to enable resolution of both haplotypes of a diploid individual.

6. A method according to claim 1 , wherein

a) the set of oligonucleotide probe members are assembled into a multifunctional detection probe, said multifunctional detection probe comprising

• at least a first allele specific nucleotide sequence, said first allele specific nucleotide sequence being specific for one allele of a first polymorphism,

• at least a second allele specific nucleotide sequence, said second allele specific nucleotide sequence being specific for one allele of a second polymorphism,

7. The method according to claim 6, further comprising using multifunctional detection probes, which can detect every combination of alleles for the multiple polymorphisms.

8. The method according to claim 1 , wherein a) one probe of the set is an allele-specific primer being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific primer being specific for one allele of a second polymorphism in said preselected region, c) isolating the target nucleic acid comprises amplifying it using the primer pair of said allele specific primers, obtaining a resolution of the haplotypes, d) detecting the presence or absence of said multiplicity of polymorphisms comprises contacting said target with a detection probe, and detecting the hybrid between the amplified target nucleic acid the detection probe.

9. The method according to claim 1 , wherein a) one probe of the set is an allele specific detection probe being specific for one allele of a first polymorphism in said preselected region, b) the other probe of the set is an allele specific detection probe being specific for one allele of a second polymorphism in said preselected region, said other probe being differently labelled, c) specifically analysing the target nucleic acid comprises contacting the target nucleic acid sequence with the probes, obtaining a resolution of the haplotypes, d) detecting the presence or absence of said multiplicity of polymorphisms comprises detecting the hybrid between the amplified target nucleic acid and the allele specific detection probes.

10. A method according to any of the preceding claims, wherein the target nucleic acid is a nucleic acid selected from the group consisting of genomic DNA, cDNA, and mRNA,

11. A method according to any of the claims 1 -7, where the target nucleic acid is a nucleic acid selected from mitochondrial DNA and chloroplast DNA.

12. A method according to any of the preceding claims, wherein at least one member of 5 the set of oligonucleotide probes is labelled.

13. A method according to claim 12, wherein said at least one member of the set of oligonucleotide probes is labelled with a fluorescent reporter group.

10 14. A method according to claim 13, wherein the fluorescent group is selected from the group consisting of fluorescein, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, acridin, Hoechst 33258, Rhodamine, Rhodamine Green, Tetramethylrhodamine, Texas Red, Cascade Blue, Oregon Green, Alexa Fluor, Tetramethylrhodamine, europium and samarium.

15 15. A method according to claim 12, wherein said at least one member of the set of oligonucleotide probes is labelled with an enzyme tag.

16. A method according to claim 15, wherein the enzyme tag is selected from the group consisting of a beta-Galactosidase, a peroxidase, horseradish peroxidase, a urease, a

20 glycosidase, alkaline phosphatase, chloramphenicol acetyltransferase and a luciferase.

17. A method according to claim 12, wherein said at least one member of the set of oligonucleotide probes is labelled with a chemiluminescent group.

25

18. A method according to claim 17, wherein the chemiluminescent group is selected from the group consisting of hydrazides such as luminol and oxalate esters.

19. A method according to claim 12, wherein said at least one member of the set of 30 oligonucleotide probes is labelled with a radioisotope.

20. A method according to claim 19, wherein the radioisotope is selected from the group consisting of ³ P, ³³P, ³⁵S, ¹²⁵l,⁴⁵Ca, ⁴C and ³H.

21. A method according to any of the preceding claims, wherein the isolation of the resolved haplotype comprises the binding of at least one member of the set of oligonucleotide probes to a solid surface.

22. A method according to claim 21 , wherein the preselected region is amplified.

23. A method according to claim 22, wherein amplification is performed by an amplification method selected from the group consisting of polymerase chain reaction (PCR), Ligase Chain Reaction (LCR), Nucleic Acid Sequence-Based Amplification (NASBA), strand displacement amplification, rolling circle amplification, and T7- polymrease amplification.

24. A method according to any of claims 1-20, wherein said preselected region of target nucleic acid is amplified by an allele specific amplification method.

25. A method according to claim 24, wherein said allele specific amplification is a primer- dependent amplification method selected from the group consisting of allele specific polymerase chain reaction (allele specific-PCR), allele specific Ligase Chain Reaction (allele specif ic-LCR), allele specific Nucleic Acid Sequence-Based Amplification (allele specif ic-NASBA), allele specific strand displacement amplification, and allele specific rolling circle amplification.

26. A method according to claim 24, further comprising:

a) the use of an allele-specific primer coupled to an entity suitable for a subsequent immobilisation reaction,

b) performing the amplification reaction, thus obtaining an amplified DNA molecule with said entity coupled to one of the DNA strands,

c) dissociating the two strands of the amplified DNA molecule,

d) binding only the coupled DNA strand from step b) to a solid surface, e) contacting the bound DNA with a multiplicity of differently labelled oligonucleotide probes each of which is capable of detecting one specific polymorphism by nucleic acid hybridisation.

27. A method according to any of claims 21-23 or 26, wherein said solid surface is selected from the group consisting of a nitrocellulose surface, a cellulose surface, a diazotized surface, and a nylon surface.

28. A method according to claim 27, wherein the binding comprises the coupling of one member of an affinity pair to the nucleic acid while the other member of said affinity pair is immobilised on the solid surface.

29. A method according to claim 28, wherein said affinity pair is an affinity pair selected from the group consisting of Biotin - streptavidin, Biotin - avidin, Digoxigenin - anti- hapten antibody, fluorescein - anti-hapten antibody, lectins - lectin receptor, Ion - Ion chelators, IgG - protein A, IgG - protein G and magnets - paramagnetic particles.

30. A method according to any of claims 21 -23 or 26, wherein the binding comprises the coupling of the nucleic acid to a photoreactive group.

31. A method according to claim 30, wherein the photoreactive group is a group selected from the group consisting of a quinone, an anthraquinone, a psoralene, a coumarin, a benzofuran, an indole and a photoactivable ketone, including benzophenone and acetophenone.

32. A method according to any of claims 21-23 or 26, wherein the binding comprises the coupling of the nucleic acid to a chemoreactive group.

33. A method according to claim 32, wherein said chemoreactive group is a group selected from the group consisting of a sulfhydryl, a primary amine and a primary phosphate.

34. A method according to any of claims 5-20, wherein the resolution of the haplotypes of a preselected region of the one member of a chromosome pair is performed by hybridisation techniques in a one-phase system.

35. A method according to claim 34, wherein said multiplicity of nucleotide polymorphisms of the preselected region is detected by hybridisation in suspension with one labelled oligonucleotide probe which is capable of detecting a multiplicity of polymorphisms, such as at least 2 polymorphisms, for example at least 3 polymorphisms, such as at least 4 polymorphisms, such as at least 5 polymorphisms, for example at least 10 polymorphisms.

36. The method according to claim 35, wherein said region is amplified.

10

37. The method according to claim 36, wherein amplification is performed by an amplification method selected from the group consisting of polymerase chain reaction (PCR), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification, rolling circle amplification, and T7

15 polymerase amplification.

38. A method according to claims 34-35, wherein said multiplicity of nucleotide polymorphisms are detected by hybridisation in suspension with a multiplicity of different labelled oligonucleotide probes each of which is capable of detecting one

20 specific polymorphism.

39. A method according to claim 36, wherein the preselected region is amplified by an allele specific amplification method according to claim 24-25.

25 40. A method according to claim 5, wherein the spacer comprises from 8 to 28 nucleotides.

41. A method according to any of the preceding claims, wherein two adjacent polymorphisms are separated by at least 15 nucleotides.

30

42. A method according to claim 41 , wherein two adjacent polymorphisms are separated by at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 100 nucleotides, more preferably at least 150 nucleotides, such as at least 200 nucleotides, for example at least 250 nucleotides, such as at least 300 nucleotides, for

35 example at least 400 nucleotides, such as at least 500 nucleotides, for example at least 750 nucleotides, such as at least 1000 nucleotides, for example at least 2000 nucleotides, such as at least 5000 nucleotides, for example at least 10000 nucleotides, such as at least 20000 nucleotides, for example at least 30 kb, such as at least 40 kb, for example at least 50 kb, such as at least 75 kb, for example at least 100 kb, such as at least 250 kb, for example at least 500 kb, such as at least 750 kb, for example at least 1000 kb.

43. A kit for detecting the presence or absence of multiple haplotypic genetic variations in a preselected region of one chromosome of a chromosome pair, comprising

44. The kit according to claim 43, wherein one probe is a capture probe coupled to an entity suitable for a subsequent immobilisation reaction and the other probe is a detection probe.

45. The kit according to claim 43, comprising a primer pair, one of which is an allele specific primer, being an oligonucleotide probe.

46. The kit according to claim 43, comprising allele specific probes to resolve all possible combinations of alleles for the two or more polymorphisms.

47. A kit for detecting the presence or absence of multiple haplotypic genetic variations in a preselected region of one chromosome of a chromosome pair, comprising

a) a multifunctional oligonucleotide probe comprising

• a first allele specific nucleotide sequence, said first allele specific nucleotide sequence being specific for one allele of a first polymorphic locus, • a second allele specific nucleotide sequence, said second allele specific nucleotide sequence being specific for one allele of a second polymorphic locus,

48. The kit according to claim 47, wherein the spacer has a length of 8 to 28 nucleotides.