WO2003018835A2 - Procede de detection rapide d'haplotypes - Google Patents

Procede de detection rapide d'haplotypes Download PDF

Info

Publication number
WO2003018835A2
WO2003018835A2 PCT/DK2002/000552 DK0200552W WO03018835A2 WO 2003018835 A2 WO2003018835 A2 WO 2003018835A2 DK 0200552 W DK0200552 W DK 0200552W WO 03018835 A2 WO03018835 A2 WO 03018835A2
Authority
WO
WIPO (PCT)
Prior art keywords
allele
nucleic acid
specific
probe
polymorphisms
Prior art date
Application number
PCT/DK2002/000552
Other languages
English (en)
Other versions
WO2003018835A3 (fr
Inventor
Mogens Fenger
Joan Bentzen
Original Assignee
Hvidovre Hospital
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hvidovre Hospital filed Critical Hvidovre Hospital
Publication of WO2003018835A2 publication Critical patent/WO2003018835A2/fr
Publication of WO2003018835A3 publication Critical patent/WO2003018835A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

  • 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.
  • 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.
  • MHC major histocompatability complex
  • 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.
  • 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.
  • SNP single nucleotide polymorphisms
  • polymorphisms in particular SNPs
  • SNPs SNPs
  • 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.
  • Mutations can be detected by specific hybridisation of oligonucleotides to the nucleic acid to be analysed.
  • 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.
  • SWNT single- walled carbon nanotube
  • AFM atomic force microscopy
  • 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.
  • 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.
  • polymorphism in the context of the present invention the term polymorphism is also intended to cover a mutation.
  • 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.
  • 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).
  • 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.
  • PCR polymerase chain reaction
  • 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
  • a "set or probe members" in intended to cover two or more probe members.
  • 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.
  • 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
  • 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
  • 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.
  • 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.
  • a) one probe of the set is an allele-specific primer being specific for one allele of a first polymorphism in said preselected region
  • 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
  • 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.
  • 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.
  • the set of oligonucleotide probe members are assembled into a multifunctional detection probe, said multifunctional detection probe comprising
  • 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.
  • target nucleotide sequence carrying a specific allele in the first locus and a specific allele in the second locus is detected.
  • a) one probe of the set is an allele-specific primer being specific for one allele of a first polymorphism in said preselected region
  • the other probe of the set is an allele specific primer being specific for one allele of a second polymorphism in said preselected region
  • 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.
  • 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.
  • one probe of the set is an allele specific detection probe being specific for one allele of a first polymorphism in said preselected region
  • 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.
  • 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.
  • one probe member for each polymorphism is needed.
  • 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.
  • 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.
  • 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
  • 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.
  • 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?
  • 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
  • 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • preselected region describes this region.
  • the size of the preselected region may vary.
  • 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.
  • polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population
  • SNPs single nucleotide polymorphisms
  • 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
  • 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.
  • 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.
  • other regions of other genes may be selected.
  • 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”.
  • 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.
  • 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 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.
  • 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.
  • chloroplast DNA may be an interesting source of target nucleic acid as is also mitochondrial DNA.
  • 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
  • Rhodamine trademarks for Biological Detection Systems, Inc.
  • fluorescein acridin, acridin orange
  • Hoechst 33258 Rhodamine
  • Rhodamine Green Tetramethylrhodamine
  • Texas Red Cascade Blue
  • Oregon Green Alexa Fluor
  • NBD 7-nitrobenzo-2-oxa-1 -diazole
  • pyrene and Europium, Ruthenium, Samarium, and other rare earth metals.
  • labelling moieties include, radioisotopes, chemiluminescent compounds, labelled binding proteins, heavy metal atoms, spectroscopic markers, magnetic labels, and linked enzymes.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase
  • 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.
  • example 2 comprises binding of the nucleic acid to a solid surface.
  • immobilisation is obtained by coupling a biotin molecule to the nucleic acid and subsequently immobilising the nucleic acid on a streptavidin modified surface.
  • 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.
  • 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.
  • 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.
  • 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.
  • the support may carry one member of an "
  • 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.
  • 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.
  • oligonucleotides may also be labelled or coupled to chemoreactive groups comprising, but not limited to: sulfhyl, primary amine or phosphate.
  • 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.
  • 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).
  • 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.
  • 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.
  • PCR polymerase chain reaction
  • 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.
  • LCR LCR for allele-specific amplification
  • NASBA Nucleic Acid Sequence-Based Amplification
  • 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.
  • 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.
  • 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.
  • hybridisation signifies hybridisation under conventional hybridising conditions, preferably under stringent conditions, as described for example in Sambrook et al., Molecular
  • 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.
  • the conditions are "highly stringent", i.e. 5-10°C under the melting point T m .
  • 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.
  • optimised hybridisation results are reached by adjusting the temperature and/or the ionic strength of the hybridisation buffer as described in the art.
  • 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).
  • PNA protein nucleic acid
  • 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).
  • 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.
  • 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".
  • 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.
  • sequences hybridising with the target nucleotide sequence are separated by a spacer sequence, which does not hybridise to the target.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 is the non-exchangeable protein of the low-density lipoprotein (LDL) complex that is recognised by the LDL-receptor.
  • LDL low-density lipoprotein
  • 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 ⁇ PCR tubes (cat.no. AB-0337, ABgene, Merck Eurolab, Denmark) containing 0.6 ⁇ M of each primer (see figure 1 , DNA-technology, Arhus, Denmark), 0.2 mM of each dNTP (Quiagen, Merck Eurolab, Denmark), 1.5 mM MgCI 2 (Applied Biosystems, Denmark).
  • 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 ⁇ L of PCR amplicon in 150 ⁇ L 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 ⁇ L Tween.
  • the surfaces are shaken at 37°C for 30 minutes and then washed with 200 ⁇ L 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.
  • 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.
  • 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 ⁇ M of the oligonucleotide in 200 ⁇ L 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 ⁇ L of SSCT buffer. The surface is then placed in a flourescens reader (Victor 2 , 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.
  • Human genomic DNA is isolated as described in examplel .
  • 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.
  • 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.
  • 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.
  • streptavidin cat.no 1741772, Roche, Denmark
  • One hybridisation is performed for each allele specific amplification.
  • An aliquot of 20 ⁇ L of PCR amplicon is mixed with 150 ⁇ L 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 ⁇ L 1 X SSCT (see example 1) and hybridised with the detection probes as follows.
  • 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 ⁇ M of each hybridisation oligo in 200 ⁇ L 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 ⁇ L Tween) is added to each tube. The tubes are shaken at 37°C for 30 minutes and then washed with 200 ⁇ L SSCT buffer 5 times.
  • 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)
  • the PCR tube is placed in a fluorescence reader (Victor 2 , 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
  • 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
  • haplotype T (in codon 2488) C (in codon 2712) will give a reading from the 596 nm filter in the tube where
  • Example 3 Detection of Apolipoprotein E (apoE) haplotypes by hybridisation to a biotinylated PCR-fragment.
  • apoE Apolipoprotein E
  • 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.
  • Human genomic DNA is isolated as described in examplel .
  • SEQ ID No. 19 and 20 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.
  • PCR amplifications are carried out in 100 ⁇ PCR tubes (ABgene, purchased from Merck Eurolab, Denmark) containing 200 ng of genomic DNA in TE buffer , 0.5 ⁇ M of each primer, 0.2 mM of each dNTP, 1.5 mM MgCI 2 , 1 U Taq-polymerase and 2,5 ⁇ L Goldbuffer, all purchased from Applied Biosystems, Denmark. Water was added to a final volume of 25 ⁇ l.
  • 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.
  • 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.
  • streptavidin catalog.no 1741772, purchased from Roche, Denmark
  • An aliquot of 20 ⁇ of PCR amplicon is mixed with 150 ⁇ 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 ⁇ 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.
  • 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 ⁇ Tween 20 is added to each tube.
  • the tubes are incubated at 37° C for 5 minutes and then washed with 300 ⁇ 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.
  • the PCR tube is now placed in a fluorescence reader (Victoi 2 , Perkin Elmer, Denmark) and readings are taken using emission filters 615 nm (for Cy3.5) and 535 nm (for Acridin orange).
  • 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:
  • 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.
  • SEQ ID Nol2 Label 3-5 ' tctGgtatatatatatatafcafctagGgtc ApoB codon 2712/2488 C/C detection probe
  • SEQ ID Nol4 Label-5' aattctAgtatgt ApoB codon 2712 T allele detection probe

Abstract

L'invention concerne un procédé et des trousses permettant de déterminer l'haplotype génétique (phase de liaison) d'une partie d'un génotype individuel, qui consiste à établir la présence ou l'absence d'au moins deux polymorphismes de nucléotides spécifiques sur l'un des deux chromosomes homologues.
PCT/DK2002/000552 2001-08-23 2002-08-22 Procede de detection rapide d'haplotypes WO2003018835A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200101252 2001-08-23
DKPA200101252 2001-08-23

Publications (2)

Publication Number Publication Date
WO2003018835A2 true WO2003018835A2 (fr) 2003-03-06
WO2003018835A3 WO2003018835A3 (fr) 2004-03-25

Family

ID=8160677

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2002/000552 WO2003018835A2 (fr) 2001-08-23 2002-08-22 Procede de detection rapide d'haplotypes

Country Status (1)

Country Link
WO (1) WO2003018835A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2008136419A (ja) * 2006-12-01 2008-06-19 Canon Inc 複対立遺伝子のハプロタイプ決定方法
EP2035578A2 (fr) * 2006-06-09 2009-03-18 Conexio 4 Pty Ltd Identification d'une molécule d'acide nucléique
EP2069497A2 (fr) * 2006-11-02 2009-06-17 University of Utah Research Foundation Oligonucleotides a utiliser en pcr specifique d'un allele
WO2017019858A1 (fr) 2015-07-30 2017-02-02 The Trustees Of The University Of Pennsylvania Allèles polymorphes de nucléotide unique de gène dp-2 humain pour la détection de la sensibilité à l'inhibition de la croissance de cheveux par pgd2
CN110050073A (zh) * 2017-03-14 2019-07-23 香港科技大学 单倍型和双倍型的确定方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992016180A2 (fr) * 1991-03-13 1992-10-01 Siska Diagnostics, Inc. Detection de la resistance a la 3'-azido-3'-desoxythymidine
WO1992019775A1 (fr) * 1991-04-29 1992-11-12 Raggio-Italgene Spa Titrage et lot d'epreuve pour le depistage des anomalies chromosomiques
WO2000020644A1 (fr) * 1998-10-06 2000-04-13 Emory University Diagnostic moleculaire de galactosemie
WO2000026412A1 (fr) * 1998-11-02 2000-05-11 Kenneth Loren Beattie Analyse d'acides nucleiques par hybridation en tandem ciblee sur des sequences
WO2000039331A1 (fr) * 1998-12-23 2000-07-06 Astrazeneca Ab Polymorphisme nucleotidique unique dans un pyruvate deshydrogenase kinase isoenzyme 2 (pdk2) chez l'homme
WO2000050869A2 (fr) * 1999-02-26 2000-08-31 Incyte Pharmaceuticals, Inc. Detection de polymorphisme nucleotidique simple (snp)
WO2000056916A2 (fr) * 1999-03-18 2000-09-28 Exiqon A/S Detection de mutations dans des genes par des amorces de lna specifiques
EP1246114A2 (fr) * 2001-03-30 2002-10-02 Perlegen Sciences, Inc. Procédés d'analyse génomique

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992016180A2 (fr) * 1991-03-13 1992-10-01 Siska Diagnostics, Inc. Detection de la resistance a la 3'-azido-3'-desoxythymidine
WO1992019775A1 (fr) * 1991-04-29 1992-11-12 Raggio-Italgene Spa Titrage et lot d'epreuve pour le depistage des anomalies chromosomiques
WO2000020644A1 (fr) * 1998-10-06 2000-04-13 Emory University Diagnostic moleculaire de galactosemie
WO2000026412A1 (fr) * 1998-11-02 2000-05-11 Kenneth Loren Beattie Analyse d'acides nucleiques par hybridation en tandem ciblee sur des sequences
WO2000039331A1 (fr) * 1998-12-23 2000-07-06 Astrazeneca Ab Polymorphisme nucleotidique unique dans un pyruvate deshydrogenase kinase isoenzyme 2 (pdk2) chez l'homme
WO2000050869A2 (fr) * 1999-02-26 2000-08-31 Incyte Pharmaceuticals, Inc. Detection de polymorphisme nucleotidique simple (snp)
WO2000056916A2 (fr) * 1999-03-18 2000-09-28 Exiqon A/S Detection de mutations dans des genes par des amorces de lna specifiques
EP1246114A2 (fr) * 2001-03-30 2002-10-02 Perlegen Sciences, Inc. Procédés d'analyse génomique

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHUBER A P ET AL: "HIGH THROUGHPUT PARALLEL ANALYSIS OF HUNDREDS OF PATIENT SAMPLES FOR MORE THAN 100 MUTATIONS IN MULTIPLE DISEASE GENES" HUMAN MOLECULAR GENETICS, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 6, no. 3, 1 March 1997 (1997-03-01), pages 337-347, XP000673446 ISSN: 0964-6906 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2035578A2 (fr) * 2006-06-09 2009-03-18 Conexio 4 Pty Ltd Identification d'une molécule d'acide nucléique
EP2035578A4 (fr) * 2006-06-09 2010-01-27 Conexio 4 Pty Ltd Identification d'une molécule d'acide nucléique
EP2069497A4 (fr) * 2006-11-02 2010-08-04 Univ Utah Res Found Oligonucleotides a utiliser en pcr specifique d'un allele
EP2069497A2 (fr) * 2006-11-02 2009-06-17 University of Utah Research Foundation Oligonucleotides a utiliser en pcr specifique d'un allele
US8580945B2 (en) 2006-11-02 2013-11-12 University Of Utah Oligonucleotides for use in allele-specific PCR
EP2099909A4 (fr) * 2006-12-01 2010-12-22 Canon Kk Procede de determination de l'haplotype de multiples genes alleliques
JP2008136419A (ja) * 2006-12-01 2008-06-19 Canon Inc 複対立遺伝子のハプロタイプ決定方法
KR101163279B1 (ko) * 2006-12-01 2012-07-05 캐논 가부시끼가이샤 다수의 대립 유전자의 하플로타입의 결정방법
EP2099909A1 (fr) * 2006-12-01 2009-09-16 Canon Kabushiki Kaisha Procede de determination de l'haplotype de multiples genes alleliques
US8637248B2 (en) 2006-12-01 2014-01-28 Canon Kabushiki Kaisha Method of determining the haplotype of multiple allelic genes
AU2007329856B2 (en) * 2006-12-01 2014-03-27 Canon Kabushiki Kaisha Method of determining the haplotype of multiple allelic genes
WO2017019858A1 (fr) 2015-07-30 2017-02-02 The Trustees Of The University Of Pennsylvania Allèles polymorphes de nucléotide unique de gène dp-2 humain pour la détection de la sensibilité à l'inhibition de la croissance de cheveux par pgd2
CN108472510A (zh) * 2015-07-30 2018-08-31 宾夕法尼亚大学理事会 用于检测pgd2对毛发生长抑制的易感性的人dp-2基因的单核苷酸多态性等位基因
CN110050073A (zh) * 2017-03-14 2019-07-23 香港科技大学 单倍型和双倍型的确定方法

Also Published As

Publication number Publication date
WO2003018835A3 (fr) 2004-03-25

Similar Documents

Publication Publication Date Title
EP0648280B1 (fr) Procede et reactif permettant de determiner des variations specifique de nucleotides
AU697642B2 (en) High throughput screening method for sequences or genetic alterations in nucleic acids
US5589330A (en) High-throughput screening method for sequence or genetic alterations in nucleic acids using elution and sequencing of complementary oligonucleotides
AU744746B2 (en) High-throughput screening method for identification of genetic mutations or disease-causing microorganisms using segmented primers
US20060199183A1 (en) Probe biochips and methods for use thereof
US20030082530A1 (en) Reagent Kit for Determining Specific Nucleotide Variations
US20050064459A1 (en) Ligation assay
US20020098484A1 (en) Method of analyzing single nucleotide polymorphisms using melting curve and restriction endonuclease digestion
US8877910B2 (en) Probe for detecting polymorphism in exon 12 of NPM1 gene and use thereof
JP4228041B2 (ja) 塩基多型の検出方法
JP2009065988A (ja) 多発性嚢胞腎症の遺伝子分析用の組成物および方法
WO2003018835A2 (fr) Procede de detection rapide d'haplotypes
US20160060696A1 (en) Method for the identification by molecular techniques of genetic variants that encode no d antigen (d-) and altered c antigen (c+w)
US20040241722A1 (en) Molecular haplotyping of genomic DNA
US20030235827A1 (en) Methods and compositions for monitoring primer extension and polymorphism detection reactions
JP2013162783A (ja) Ｐｏｎ１遺伝子多型（ｑ１９２ｒ）を検出する方法
JP5530185B2 (ja) 核酸検出方法及び核酸検出用キット
KR102451403B1 (ko) ＡｐｏＥ 유전자의 ＳＮＰ 분석을 위한 실시간 검출용 단일핵산 및 이를 이용한 검출 방법
US20230098408A1 (en) Single nucleic acid for real-time detection for snp analysis of apoe gene and detection method using the same
JP4650420B2 (ja) 塩基判定方法及び塩基判定用キット
Aydin et al. Validation of fluorescence-labeled artificial nonhuman sequences for single-strand conformation polymorphism mutation detection in familial hypercholesterolemia
JP2005245272A (ja) アルコール脱水素酵素遺伝子多型の簡易検出方法および検出用試薬
CN115873926A (zh) 用于分析ApoE基因的SNP的实时检测用单核酸及利用其的检测方法
van Himbergen et al. High-throughput genotyping with infrared Fluorescence Allele Specific Hybridization (iFLASH): a simple, reliable and low-cost alternative
JP2010246400A (ja) 多型の識別方法

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GM HR HU ID IL IN IS JP KE KG KP KZ LC LK LR LS LT LU LV MA MD MK MN MW MX MZ NO NZ OM PH PT RO RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VC VN YU ZA ZM

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ CZ DE DE DK DK DM DZ EC EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SK SL TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR IE IT LU MC NL PT SE SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP