WO1992018646A1 - Sondes et amorces de nucleotides pour analyses genetiques - Google Patents

Sondes et amorces de nucleotides pour analyses genetiques Download PDF

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WO1992018646A1
WO1992018646A1 PCT/GB1992/000709 GB9200709W WO9218646A1 WO 1992018646 A1 WO1992018646 A1 WO 1992018646A1 GB 9200709 W GB9200709 W GB 9200709W WO 9218646 A1 WO9218646 A1 WO 9218646A1
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sequence
dna
polynucleotide
probes
oligo
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PCT/GB1992/000709
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Peter Gill
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The Solicitor For The Affairs Of Her Majesty's Treasury
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    • 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
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to poly- and oligonucleotides, particularly to DNA and RNA probes and polymerase chain reaction (PCR) primers, methods for their production and uses for the probes and primers in restriction fragment length polymorphism (RPLP) analysis and minisatellite variant repeat mapping (MVRM) analysis respectively.
  • PCR polymerase chain reaction
  • RPLP restriction fragment length polymorphism
  • MVRM minisatellite variant repeat mapping
  • hypervariable DNA In complex organisms such as man a large proportion of the DNA in the genome does not appear to encode protein but has an as yet unknown function. A characteristic of this DNA, in contrast to that which encodes protein, is that it varies between individuals of any one species, eg, between different human beings, and is hence termed hypervariable DNA.
  • This hypervariable DNA contains a considerable amount of repeat sequences, either dispersed throughout the genome or arranged in long arrays of short tandem repeat loci.
  • the tandem repeat units known as 'minisatellites' are often highly polymorphic due to variation in the number of copies in the repeat units.
  • the polymorphism of these minisatellites appears to be unique to individuals of one species to the extent that the possibility of two individuals having the same polymorphism pattern is around 1 in 10 9 ; that figure excluding monozygotic twins.
  • analysis of human DNA hypervariable minisatellite poymorphism patterns has been made possible by the development of DNA probes which are complementary to, and hence which hybridize with, these repeat sequences and which enable the pattern of polymorphism to be visualised after restriction cleavage by the use of radiolabelled probes and autoradiography.
  • This method of genetic characterisation has been a powerful tool in a number of applications including paternity testing and so called f DNA fingerprinting' (see EP 0186271 and EP 0238329 for example of such use) .
  • the latter technique typically involves the forensic positive association of individuals with bodily residues such as blood, hair, semen, etc found at the locus of an incident under investigation and involves the use of such probes as that described in GB 2166445 A.
  • DNA probes for these minisatellites are generally of two main types, multilocus probes and single locus probes (the latter also being referred to as locus specific probes) .
  • Multilocus probes such as that of GB 2166445 A, are complementary to and hence recognative of minisatellites that occur at more than one locus throughout the genome and tend to produce complex autoradiographic patterns.
  • Single locus probes hybridize to a sequence which is only found in one position on the genome. The single locus probes therefore produce much simpler autoradiographic patterns than the multilocus probes and thus results are correspondingly easier to interpret but do not provide as much information, having probabilities of random identity of the order of 1 in 10,000 or so.
  • a further technique in which minisatellite regions may be analysed is by polymerase chain reaction (PCR) amplification of target sequences and analysis of the oligonucleotide products obtained by separation using gel chromatography and/or ability to hybridize with characteristic probe sequences using such techniques as Southern blotting.
  • Oligonucleotide sequences disclosed herein as occurring in genomic DNA can be targeted by pairs of oligonucleotide primers, themselves consisting of short oligonucleotides of the present invention and the PCR technique can be used to produce charateristic sequences of varying length in the now well known manner (eg. see J. Nucleic Acids Res. __£ 10953-10971. 1988; Nature Vol 352, 427 ⁇ 9. 1991.
  • a particularly preferred form of the PCR technique is known as minisatellite variant repeat mapping (MVRM or MVR-PCR) and uses one primer (at least) which is targeted at a sequence in DNA flanking the hypervariable loci and one (at least) targeted within the hypervariable loci (see Jeffreys et al; Nature Vol 354, 21 November 1991) '
  • the primer targeted at the sequence within the loci comprises a sequence capable of specific hybridization with the relevant repeat unit and in preferred form further has a 5' sequence of bases which are not capable of specific binding. In this way the amount of 'internal' amplification of DNA, wherein shorter and shorter PCR products are derived from PCR repeat products, is reduced and inter alia a more reliable result obtained.
  • Oligonucleotides of the present invention which have such non-specific 5' sequence thus find application as primers targeted at hypervarible regions for use in this preferred MVR-PCR technique.
  • the present inventor has identified a repeat sequence that occurs in hypervariable loci of human genomic DNA and has determined that this sequence is suitable for use as a target for application of genetic analysis techniques such as RFLP and MVRM.
  • the present invention may be readily defined in terms of oligo- and polynucleotide sequences which are capable of specifically hybridizing to that sequence of its complementary sequence for either probe or primer use.
  • the present invention provides a DNA or RNA oligo- or polynucleotides, in isolated, vectored, cloned and/or labelled form, having a nucleotide base sequence capable of specific hybridization under Southern Blot hybridization conditions with a target nucleotide sequence I or its complementary sequence II:
  • L represents T or C
  • M represents A or G
  • N represents G or T
  • Q represents C or A
  • n is an integer from 1 to 1000 and 5' and 3' refer to the respective 5' an ⁇ ' 3' ends of the sequence, wherein A, G, C and T relate to bases adenine, guanine, cytosine and thymine/uracil respectively.
  • the present invention provides a DNA or RNA oligonucleotide as described above characterised in that it is capable of functioning as polymerase chain reaction primer for specific amplification of a sequence I and/or II when used together with a second such primer or a primer selected to target a region outside a genomic hypervariable loci comprising said sequence I or II.
  • RNA oligo- or polynucleotides in isolated, vectored, cloned and/or labelled form having a nucleotide base sequence which comprises a sequence I:
  • n is an integer from 1 to 1000 and 5' and 3' refer to the respective 5' and 3' ends of the sequence or sequences having up to 1 % mismatch to them.
  • sequences I and II may comprise the entire sequence or may be flanked by further such base sequences; no particular limit is placed on the length of flanking sequences connected to I and II other than by factors commonly taken into account, ie; handling and manipulating properties associated with use for hybridization.
  • the symbols C, A, G and T refer to the bases cytosine, adenine, guanine and thymine and to analogous bases having equivalent base pairing characteristics to each base respectively.
  • the symbol T also relates to the base uracil which as well as having equivalent base pairing characteristics to thymine, is the usual base found in its stead in RNA sequences.
  • the invention also provides oligo- or polynucleotides comprising sequences I or II wherein one or more of the bases shown above have been replaced by a base which has equivalent base pairing characteristics such as those where thymine or uracil is replaced by inosine or those where guanine is replaced by 8-azaguanine as will be known to the man skilled in the art.
  • the order of the symbols indicates the sequence in which they occur along the nucleotide backbone.
  • 15% mismatch it is meant that 15% of the bases are other than those specified for a given point in the sequence. Lest this appear to be vague, it will be understood by the man skilled in the art that such sequences may still be effective at hybridizing at reasonable specificity to a target sequence as long as lower stringency hybridization conditions are employed, ie. by reducing the temperature used when melting target DNA and combining it with the probe. Lower stringency matched sequences may also have utility in so far as they are capable of producing characteristic gel patterns.
  • sequences may comprise a single sequence I or II or from two (2) to several hundred (-00) tandem repeat sequences comprising the sequences I to II, more preferably comprising between 100 and 800, most preferably between 200 and 300 tandem repeats.
  • n 1
  • MVRM MVRM
  • primers will consist of the sequences targeted at the genomically occurring sequences I and/or II which comprise sequences complementary to all or a specific part of the sequence I or II repeat unit contiguous with a non-specific (thus non-hybridizing at hybridiztion conditions used) sequence in a manner analogous to that described in the Nature Vol 354 reference above.
  • Particularly preferred target, primer or probe sequences are those where the sequence I or II comprises one or more of the sequences:
  • nucleotides of the invention comprise the tandemly repeated sequences, some of these repeats may be imperfect repeats. Probe sequences of the invention may also be combined with flanking sequences, but the nature of these is largely irrelevant and thus they are not essential. The presence of larger flanking sequences may be useful as used as carriers from which smaller oligo- and polynucleotides containing the essential sequences may be excised; the term essential sequences referring to any of those falling into the definition of sequences I, or II as defined above.
  • sequences of the invention may conveniently be prepared by any of the known methods of oligo- or polynucleotide synthesis, eg; by chemical coupling of individual bases; by polymerase chain reaction (PCR) or by recombinant DNA transformation of cells and their subsequent culture.
  • the sequences are converted into probes by labelling them in any way which allows their identification by convenient means, ie. by attaching a characteristic entity to the sequence which has a property which will distinguish it from other, non-labelled sequences.
  • the sequence when the sequence is comprised of several hundred repeats it may be derived from a clone product of said recombinant DNA technique whereby a known sequence I or II is inserted into the DNA of a host cell using a suitable vector in the known way and the transformant cell is cultured to produce an increased source of the desired sequence.
  • the present inventors have provided a recombinant cell line having a sequence according to the invention incorporated within its DNA and which they have deposited at the European Collection of Animal Cell Cultures, PHLS, CAMR, Porton Down, Salisbury, Wiltshire, United Kingdom on 15th March 1991 under the terms and conditions of the Budapest Treaty, bearing deposition number 91031501; these cells and their use as a source of poly- and oligonucleotide material of the invention being a further aspect of the invention and providing the basis for further mutations and transformants which may be used to provide alternative sources of probe or primer material.
  • the probes and primers of the invention are capable of hybridizing with fragments of DNA containing a minisatellite from a single specific minisatellite region or hypervariable locus and as such are locus specific.
  • locus specific is used herein in relation to a primer or probe to mean that the primer or probe may be used under hybridization stringency conditions which ensure that it hybridizes to only a single specific locus, eg. by control of temperature as known.
  • the locus specific probes of the present invention when used at reduced hybridization stringencies may be capable of differentiating DNA by reference to more than one polymorphic minisatellite region or hypervariable locus and may thus be used to identify other informative genetic loci.
  • the degree of mismatch which will still enable hybridization with target DNA at a given temperature may be approximately calculated for a given length of probe using various equations (Wallace et al, (1979). Nucleic Acids Res, 6, 35 3 ⁇ 366, or Membrane transfer and detection methods, pub. by Amersham International).
  • Nucleic Acids Res, 6, 35 3 ⁇ 366, or Membrane transfer and detection methods pub. by Amersham International
  • T d °C 8l.5°C 16.6 log M + 0Al(%Q+%G) - 500/n -0.6l(*formamide)
  • T d is the melting point of the target DNA duplex.
  • A, T, G and C are the number of those bases, adenine, thiamine, guanine and cytosine (or their equivalents as given above) in the probe sequence and n is the number of base pairs in the probe, may be used to determine the temperature at which probing with a probe of given composition will result in hybridization. In the absence of formamide the equation may still be used by ommision of the formamide factor. For single stranded probes longer than 100 nucleotides the melting temperature required decreases by 1°C for every 1% of mismatched base pairs. Thus mismatched probes or other loci can be worked with.
  • a method of characterising a test sample of genomic DNA with reference to one or more controls comprises fragmenting sample DNA with one or more restriction enzymes which do not cleave a sequence corresponding to a tandem repeat to any significant extent, probing the DNA fragments by hybridization with a probe of the invention, detecting fragments of DNA which have hybridized with the probe and comparing the hybridized fragments with said control or controls.
  • a preferred way of detecting the hybridized fragments is by the use of an oligo- or polynucleotide of the invention which has been chemically, biologically or radioactively labelled, preferably radioactively.
  • a preferred radiolabel is 32 P. This may be attached to the probe by esterification of the ' terminal hydroxyl group, eg, using [ - 32 P] ATP.
  • Hybridized fragments may then be visualised by autoradiography according to well known technical principles (eg. see Gill et al, (1990), Hum. Genet. 85, 75 ⁇ 79).
  • the probes of the invention may be used individually or in conjunction with other polynucleotide probes, either simultaneously in a pool of mixed probes or sequentially with other locus specific probes.
  • the probes and primers of the invention may be useful with regard to the following areas of human genetics technology: paternity and maternity testing, family group verification, zygosity testing, inbreeding testing, animal and plant pedigree analysis, genetic disease loci identification, cell chimaerism studies Cell chimaerisation studies allow donor/recipient cells to be monitored after transplant. Further use might be made in the field of analysis of tumour cells and tumours for the existence of molecular abnormalities or for testing the stability and purity of cell lines.
  • the probes and primers of the present invention are particularly useful for characterising human genomic DNA in forensic medicine and more particularly in fingerprinting semen, blood, hair, skin and/or other tissue samples in rape, homicide and similar investigations.
  • the probes and primers of the invention are mixed with samples of DNA, for example such samples as extracted by known methods from a forensic sample such as those referred to above from a victim, the scene of a crime or from a suspect.
  • the DNA samples are cleaved using a restriction enzyme to produce smaller fragments some of which contain minisatellite regions including sequences to which the probe of the invention is capable of hybridizing. These fragments of DNA are then sorted on the basis of size, preferably by gel-electrophoresis. After sorting the fragments are denatured into single stranded DNA, eg, by heating. The fragments are then immobilised by transfer onto a support, preferably by the Southern blot transfer technique using a solid nitrocellulose or nylon support membrane. When the fragments have been immobilised the support plus fragments is exposed to the labelled, eg, radiolabelled, probe of the invention by the immersion in a solution containing the probe whereupon the probe hybridize to fragments which include DNA complementary to it.
  • the hybridized fragments may be visualised by placing an X-ray film in direct contact with the support so that a pattern of bands of exposed film are produced in the positions corresponding to them.
  • a pattern of bands of exposed film are produced in the positions corresponding to them.
  • MVRM For use with PCR and particularly MVRM intact genomic DNA can be used (see Nature Vol 35 but digested DNA may be preferable if speed is not important. Primers selected from suitably specific ones targeted at the repeat sequences I to X may be used optionally paired with a primer targeted outside, but preferably closely adjacent to, the hypervariable region. The primers will routinely be unlabelled and the PCR products will be identifiable, inter alia, by standard techniques such as by SDS gel electrophoresis and Southern blotting. For MVRM the primers will preferably comprise non-hybridizing sequence flanking the hybridizing sequence in the PCR non-extending direction.
  • Fig 1 shows the distribution bands of various alleles as visualized using the probe B6.7 (as described herein), to hybridize against genomic DNA isolated from 28 asian human samples. The frequency ⁇ % ) of occurrence of each of the HinFI restriction produced bands is given against the size of said bands (kilobases).
  • EXAMPLE 1 DNA synthesizer preparation of oligonucleotides:
  • Oligonucleotides were synthesized on an ABI (Applied Biosysterns Ltd, 7 Kingland Grange, Warrington, WA14SR) automated DNA/RNA synthesizer Model 381A using beta-cyanoethyl diisopropyl phosphoramidite (CE-phosphoramidite) monomers and chemistry (Gait, M.J. (Editor) Oligonucleotide synthesis-a practical approach, IRL Press Oxford) , 1984) based on a literature method (Beaucage and Caruthers, (1981) Tetrahedron Letters, I ⁇ 59 ⁇ l862) using dimethylaminophosphoramidites. All reagents are commercially available (eg. from ABI).
  • the 3' terminal nucleoside of the sequence is attached to a controlled pore size silica gel (CPG) by an organic spacer arm.
  • CPG controlled pore size silica gel
  • An appropriate amount of this solid phase carrier is supplied in a plastic column to place on the machine to give synthesis at the 0.2, 1.0 or 10 micromolar level according to the protocol supplied by ABI (Model 38IA DNA Synthesizer User's Manual). Synthesis proceeds from 3' to 5' ends.
  • Aminolinker 2 reagent (ABI Part No 400808) can be entered as an X base at the 5' terminus of the sequence and is automatically coupled by the same reaction as the normal phosphoramidites.
  • the CPG is treated initially at room temperature with 35% v/v ammonia in water to detach the oligonucleotide by hydrolysis of the 3' link of the organic spacer molecule.
  • This treatment with ammonia is continued overnight at 55°C in sealed vials to remove various residual protective groups from the phosphate linkages and the nucleotide bases to give a biologically active oligonucleotide.
  • an Aminolinker 2 has been coupled this deprotects an active amino residue for attachment of the above mentioned markers.
  • the product is purified by 3M sodium acetate/ethanol precipitation directly from the ammonia solution in the ratio 360:40:1200 for oligo:acetate:absolute ethanol respectively.
  • a charomid library was prepared from genomic DNA pooled from 20 unrelated individuals. Human DNA was fully digested with SauIIIa and the 6-9kB fragments were purified using electrodialysis as previously described (Armour et al (1990) Genomics, 8, 501-512). 150ng DNA from the 6-9kB fraction was ligated into the BAMHI site of charomid 9 ⁇ 3 (Saito and Stark, 1986) . The charomid was packaged using Gigapack Gold and transferred into NM554 cells (recA mcrA, mcrB, ) (Raleigh et al, (1988) Nucleic Acids Res, 16, 1563-1575).
  • the cells were plated onto LUB/Ampicillin/agar and grown overnight. Hybond N filters were placed on each agar plate and used for lifting colonies from agar plates. The DNA on the filters was subjected to denaturation and fixed by microwave treatment (Buluwela et al, (1989) Nucleic acids Res. 17, 452).
  • Probe 3'HVR alpha globin was labelled with 32 P by random oligonucleotide priming (Feinburg and Vogelstein, (1984) Anal. Biochem. 137, 266-267) . Filters were incubated overnight at 6l°C in hybridization solution (Church and Gilbert, (1984), Proc. Natl. Acad. Sci. USA. 81, 1991-1995) containing 0.5ng/ml of labelled probe, without competitor DNA. Filters were washed under low stringency conditions in IxSSC, 0.1#3DS at 6l°C. Autoradiographs of the filters were prepared using Amersham-MP film. Colonies which showed signals were picked and replated at low density; filters were prepared and autoradiographed as previously described.
  • a 7-8kB fragment, designated B6.7 was purified using the above procedure and approximately 90 micrograms were prepared for further experimentation and sequencing.
  • the deposited cell line 9103101 was produced by ligation of B6.7 into pUCl ⁇ and subsequent transfection of gram negative E. coli strain DH5alpha F 1 cells with that plasmid.
  • EXAMPLE Derivation of polynucleotide B6.7 from cells of deposited cell line B6.7-Accession No. 91031501.
  • Recombinant plasmid DNA was prepared by alkaline lysis (ibid) and purified on caesium chloride-ethidium bromide density gradients. B6.7 insert DNA was isolated by digestion of recombinant plasmid with the restriction enzyme SauIIIa, electrophoresed on an agarose gel, electroeluted and purified by standard methods (ibid) .

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Abstract

Cette invention concerne des poly- et oligonucléotides, surtout les sondes d'ADN et de ARN ainsi que des amorces de réaction en chaîne de polymérase (PCR), leurs méthodes de production et leurs utilisations dans le polymorphisme de longueurs de fragments de restriction (RPLP) et l'analyse de localisation à répétition par variation de minisatellite (MVRM). Les sondes de l'invention ont une application spécifique à titre de sondes de locus simple utilisables dans les techniques de caractérisation génétique. Les sondes et amorces ont des séquences à base de nucléotides capables d'une hybridation spécifique avec une séquence comprenant une séquence I: 5' (CCCCT CACTG TCCAC (L)CTC(M) TG(N)CC TATAG AGAC)n 3', ou sa séquence complémentaire II: 3' (GGGGA GTGAC AGGTG (M)GAG(L) AC(Q)GG ATATC TCTG)n 5' ou des séquences ayant une non-correspondance avec celles-ci allant jusqu'à 15 %; où L représente T ou C; M représente A ou G; N représente G ou T; et Q représente C ou A; n est un nombre entier de 1 à 1000, et 5' et 3' désignent les extrémités 5' et 3' de la séquence. T est thymine ou uracile et les bases peuvent être remplacées par celles dotées de caractéristiques équivalentes d'appariage de bases.
PCT/GB1992/000709 1991-04-18 1992-04-16 Sondes et amorces de nucleotides pour analyses genetiques WO1992018646A1 (fr)

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Cited By (5)

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WO1995027077A1 (fr) * 1994-03-31 1995-10-12 University Of Leicester Minisatellite du chromosome y
US5811235A (en) * 1991-08-27 1998-09-22 Zeneca Limited Method of characterisation
US5853989A (en) * 1991-08-27 1998-12-29 Zeneca Limited Method of characterisation of genomic DNA
WO1999066070A1 (fr) * 1998-06-18 1999-12-23 Tomaras Constantine Biometrie adn utilisee aux fins d'identification
US6500616B1 (en) 1998-04-16 2002-12-31 Case Western Reserve University Methods of monitoring genomic integrity and detecting genomic destabilization of plant cells in tissue culture

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WO1989011547A1 (fr) * 1988-05-20 1989-11-30 Cetus Corporation Procede de determination de type de hla dp
GB2221910A (en) * 1988-08-17 1990-02-21 Sapporo Breweries DNA encoding a uricase gene
EP0370719A2 (fr) * 1988-11-25 1990-05-30 Imperial Chemical Industries Plc Des séquences nucléotidiques élongées
EP0416817A2 (fr) * 1989-09-06 1991-03-13 Zeneca Limited Procédé d'amplification

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WO1989007658A1 (fr) * 1988-02-18 1989-08-24 University Of Utah Identification genetique avec des adn d'investigation ayant des sites repetitifs en tandem en quantite variable
WO1989011547A1 (fr) * 1988-05-20 1989-11-30 Cetus Corporation Procede de determination de type de hla dp
GB2221910A (en) * 1988-08-17 1990-02-21 Sapporo Breweries DNA encoding a uricase gene
EP0370719A2 (fr) * 1988-11-25 1990-05-30 Imperial Chemical Industries Plc Des séquences nucléotidiques élongées
EP0416817A2 (fr) * 1989-09-06 1991-03-13 Zeneca Limited Procédé d'amplification

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NATURE vol. 354, no. 6350, 21 November 1991, LONDON GB pages 204 - 209; A. JEFFREYS ET AL: 'MINISATELLITE REPEAT CODING AS A DIGITAL APPROACH TO DNA TYPING' *

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US5853989A (en) * 1991-08-27 1998-12-29 Zeneca Limited Method of characterisation of genomic DNA
WO1995027077A1 (fr) * 1994-03-31 1995-10-12 University Of Leicester Minisatellite du chromosome y
US6500616B1 (en) 1998-04-16 2002-12-31 Case Western Reserve University Methods of monitoring genomic integrity and detecting genomic destabilization of plant cells in tissue culture
US6773889B2 (en) 1998-04-16 2004-08-10 Case Western Reserve University Method for detecting genomic destabilization arising during tissue culture of plant cells
WO1999066070A1 (fr) * 1998-06-18 1999-12-23 Tomaras Constantine Biometrie adn utilisee aux fins d'identification

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