EP1002127A1 - Methode de determination du genotype d'un organisme par utilisation d'une sonde d'oligonucleotides specifique a l'allele s'hybridant a des regions flanquantes de microsatellites - Google Patents

Methode de determination du genotype d'un organisme par utilisation d'une sonde d'oligonucleotides specifique a l'allele s'hybridant a des regions flanquantes de microsatellites

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EP1002127A1
EP1002127A1 EP98932341A EP98932341A EP1002127A1 EP 1002127 A1 EP1002127 A1 EP 1002127A1 EP 98932341 A EP98932341 A EP 98932341A EP 98932341 A EP98932341 A EP 98932341A EP 1002127 A1 EP1002127 A1 EP 1002127A1
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Prior art keywords
sequence
aso
microsatellite
target dna
probe
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German (de)
English (en)
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Keith Joseph IACR-Long Ashton Res Station EDWARDS
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University of Bristol
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University of Bristol
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Priority claimed from GB9714005A external-priority patent/GB9714005D0/en
Priority claimed from GBGB9806702.8A external-priority patent/GB9806702D0/en
Application filed by University of Bristol filed Critical University of Bristol
Publication of EP1002127A1 publication Critical patent/EP1002127A1/fr
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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
    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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 a method of determining the genotype of an organism using an allele specific oligonucleotide (ASO) probe and to a method of designing an ASO probe.
  • ASO allele specific oligonucleotide
  • Marker assisted breeding relies upon polymorphic genetic markers.
  • markers include restriction fragment length polymorphisms (RFLPs) (Edwards, M.D. et al , Theor. Appl. Genet. 83, 165-714 (1992)) amplified fragment length polymorphisms (AFLPs) (Vos, P et al. , Nucleic Acids Research 23, 4407-4414 (1995)) and simple sequence repeats (SSRs) or microsatellite loci (Weber, J.L. and May, P.E. , American Journal of Human Genetics 44, 388-396 (1989)).
  • RFLPs restriction fragment length polymorphisms
  • AFLPs amplified fragment length polymorphisms
  • SSRs simple sequence repeats
  • microsatellite loci Weber, J.L. and May, P.E. , American Journal of Human Genetics 44, 388-396 (1989)
  • the methods for detecting these polymorphic markers all rely upon electrophoretic separation of DNA in inert gels (agarose, acrylamide). For example, at microsatellite loci, the variation in allele lengths arising from differences in the number of repeat units, can be detected by a combination of polymerase chain reaction (PCR) amplification and polyacrylamide gel electrophoresis . Developments in fluorescent DNA fragment analysis not only make it possible to analyse many SSR loci simultaneously but also to automatically capture the data electronically (Ziegle, J. et al. , Genomics 14, 1026- 1031 (1992)). Despite the advent of these semi-automated systems or refinements such as capillary gel electrophoresis, gel-based technology is very labour intensive and time consuming for the large scale genotyping required both in experimental genome analysis and in marker assisted breeding programmes.
  • inert gels agarose, acrylamide
  • Microsatellite markers are currently a favoured marker for genotyping. They are single locus, co-dominant and multi-allelic and they are based upon the PCR which is relatively cheap to perform and can be automated. Current technology relies on the variability that exists within the simple sequence repeat copy number, i.e. one genotype may have 20 CT repeats at a specific loci whilst another may have 21 CT repeats. This difference can be detected by gel electrophoresis of the PCR products generated by using PCR primers which flank the simple sequence repeat. This is the main problem with SSRs; gel electrophoresis is time consuming and expensive to perform. The ideal genotyping test would not employ gel electrophoresis.
  • ASO Allele Specific Oligonucleotides
  • ASOs have the potential to be a quick, cheap, multiallelic and multi-locus test, they should be in regular use within genotyping laboratories. Unfortunately, whilst they are in regular use for the detection of certain human genetic diseases, they are not in regular use for non-human genotyping. The reason for this becomes apparent when one considers the enormous cost of developing ASOs. For each locus, a mapped single copy probe has to be sequenced and suitable PCR primers designed. These primer must then be used to amplify the corresponding fragment from all the other possible genotypes. These fragments must then sequenced and the sequences compared with one another to determine ASOs for each of the possible alleles. In addition, when one considers that in an average plant genotyping laboratory, 100 different loci might routinely be screened, then the amount of work required to develop ASOs for each locus and each possible allele, becomes considerable.
  • a recurrent problem with microsatellite markers in pigs and other animals is the incidence of so-called "null” or pseudo-null alleles. These null alleles are revealed as repeated failure to produce a PCR product and are attributed to polymorphisms at the primer binding site preventing primer annealing.
  • 50 out of 400 porcine microsatellites were recorded as exhibiting null alleles.
  • the variant nucleotide is likely to be located within 5 nucleotides of the 3' end of the primer.
  • each primer pair effectively assays 10 nucleotide positions for polymorphisms. It is possible to predict the frequency of polymorphisms from the observations of Alexander et al. , ⁇ Animal Genetics 27, 137-148 (1996)) as 50 per 400 x 10 nucleotides i.e. 1 per 80 nucleotides which is 3 to 4 times the level expected in random mammalian genomic DNA.
  • a method of determining the genotype of an organism comprising the step of hybridising an Allele Specific Oligonucleotide (ASO) probe to a target DNA sequence, wherein the target DNA sequence comprises a sequence of DNA which flanks a microsatellite repeat unit or Simple Sequence Repeat (SSR).
  • ASO Allele Specific Oligonucleotide
  • SSR Simple Sequence Repeat
  • the genotype of an organism can be defined as the genetic constitution of an individual organism, as distinct from its phenotype which is the total appearance of an organism determined by interaction during development between its genetic constitution and the environment. Different phenotypes may result from identical genotypes, but is generally unlikely that two organisms could share all their phenotypic characters without having identical genotypes.
  • the present invention is applicable to all organisms, particularly plants, animals and fungi, including yeasts. Methods of the present invention may find utility to species of non- flowering and flowering plants, both monocotyledonous and dicotyledenous. Plant species of interest include, but are not limited to maize ⁇ Zea mays), teosinte, Arabidopsis thaliana, Brassica spp. , cereals (e.g. oats, barley, wheat, rye), banana, palms, ornamental plants (e.g. orchids, lilies, tulips, roses, clematis), trees (e.g.
  • Yeast species include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pischia, Kluyveromyces lactis.
  • Fungal species include, but are not limited to both pathogenic and non-pathogenic fungi, for example the wheat fungal pathogen Septoria.
  • the invention is also applicable to all animals, including birds, such as domestic fowl, amphibian species, reptile species and fish species. In practice, however, it will be to animals, especially marsupials or mammals, particularly placental mammals that the greatest commercially useful application is presently envisaged.
  • mice may find general application to humans and also to non-human animals, preferably mammals. It is with ungulates such as cattle, sheep, goats, water buffalo, camels and pigs that the invention is likely to most useful. It should also be noted that the invention is likely to be applicable to other economically important animal species such as, for example, horses, llamas, alpacas or rodents, e.g. mice, rats or rodents.
  • transgenic should not be taken to be limited to referring to an organism as defined above containing in their germ line one or more genes from another species, although many such organisms will contain such a gene or genes. Rather, the term refers more broadly to any organism whose germ line has been the subject of technical intervention by recombinant DNA technology.
  • Hybridisation of an oligonucleotide probe to a target DNA sequence in a method according to the present invention includes the denaturation of a source of duplex DNA, also known as "melting" , in which the DNA is allowed to anneal with an appropriate oligonucleotide probe under certain conditions. Denaturation of double- stranded DNA (dsDNA) may be achieved by small increases in the temperature of DNA in solution.
  • dsDNA double- stranded DNA
  • highly stringent conditions means hybridisation to DNA bound to a solid support in 0.5M NaHPO 4 , 7% sodium dodecyl sulfate (SDS), lmM EDTA at 65°C, and washing in O. lxSSC/0.1 % SDS at 68°C (Ausubel et al eds. " Current Protocols in Molecular Biology” 1, page 2.10.3, published by Green Publishing Associates, Inc. and John Wiley & Sons, Inc. New York (1989)). In some circumstances, less stringent hybridisation conditions may be required.
  • hybridisation conditions means washing in 0.2xSSC/0.1 % SDS at 42°C (Ausubel et al (1989) supra).
  • Hybridisation conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex.
  • convenient hybridisation temperatures in the presence of 50% formamide are: 42°C for a probe which is 95 to 100% homologous to the target DNA, 37°C for 90 to 95% homology, and 32°C for 70 to 90% homology.
  • the complexity of the hybridisation reaction carried out depends upon the length of the sequence on the array and the application (reviewed in Marshall, A. and Hodgson, J., Nature Biotechnology 16 27-31 (1998)).
  • a set of four oligonucleotides is generally designed (one for each base type) that spans each position in the target sequence, differing only in the identity of the central base. The relative intensity of hybridisation to each series of probes at a particular location identifies the base.
  • Each set of oligonucleotides is offset by one base so that they can be arranged in order by analysing overlaps, a process known as "tiling" . If the application is expression monitoring, where details of the precise sequence are unnecessary, sets of oligonucleotides are constructed that identify unique motifs in genes. By arranging them in a particular order, it is possible to identify chromosomal location as well as sequence. In contrast, arrays of cDNA work more like conventional dot-blots where competitive hybridisation of two labelled samples (disease versus normal; heat-shock induced versus normal) reveals different gene expression.
  • Allele specific oligonucleotides (ASOs) in accordance with the present invention may comprise oligonucleotide or short polynucleotide sequences of deoxyribonucleotides known as "bases" which typically include one or more of deoxyriboadenosine (A), deoxyribocytosine (C), deoxyriboguanosine (G) and deoxyribothymosine (T).
  • Other possible component bases include deoxyriboinosine (I) and/or chemically-modified variants A, C, G, T, or I, for example methylated derivatives.
  • the ASO probe sequence may be from 10 to 50 bases long, suitably 12 to 15 bases, preferably 15 to 21 bases.
  • the ASO probe may be comprise a peptide nucleic acid (PNA) (Nielsen et al Science 254 1497- (1991); Eghom et al J. Am. Chem. Soc. 114 1895- (1992); Llanvey et al Science 258 1481- (1992)).
  • PNAs are nucleic acid analogues composed of a polymer of 2-aminoethyl glycine which acts as the backbone of the molecule. Each monomer is linked by a methylenecarbonyl linkage to one of the bases found in DNA or RNA.
  • the use of a PNA molecule as the ASO has the effect of increasing the stability of the correct hybridisation versus the mismatch hybridisation.
  • the target DNA sequence is a short region of DNA in the DNA of the organism to be investigated, in which the target sequence flanks a microsatellite repeat unit.
  • the DNA sequence may be an oligonucleotide or a polynucleotide sequence as appropriate, of from 5 to 500 base pairs, suitably of from 10 to 100 base pairs, preferably, 15 to 45 base pairs.
  • a microsatellite repeat unit or Simple Sequence Repeat (SSR) is a marker in a DNA sequence which is single locus, co-dominant and multi-allelic. SSRs are short tandem repetitive DNA sequences with a repeat length of a few (1 to 5) base pairs.
  • the repeat unit may be any base, e.g., A, G, C, or T, or any pair of bases, e.g. CA, CT, AT, etc.
  • the SSR may be repeated up to around 8 to 10 repeat units roughly every 10-20 kilobases in the genome of an Organism. Higher number repeat unit units of up to 100 repeats are often called “minisatellite” DNA regions and beyond 100 repeat units the terminology is generally "satellite" DNA regions.
  • Methods in accordance with the present invention to determine the genotype of an organism can also suitably make use of the Polymerase Chain Reaction (PCR) to identify the hybridisation of an ASO to a region flanking a microsatellite region of interest.
  • PCR can be carried out, e.g. by use of a Perkin-Elmer / Cetus thermal cycler with Taq polymerase (Gene AmpTM) or Taq Gold polymerase as described in Erlich et al ⁇ Nature 331 461-462 (1988)).
  • ASA allele-specific amplification
  • ASA ASA
  • DNA regions i.e. several mutations
  • Some oligonucleotides carry the mutation in the centre of the molecule so that differential amplification depends on differential hybridisation
  • COP competitive oligonucleotide priming
  • amplification-refractory mutation system abbreviated as amplification-refractory mutation system or ARMS.
  • Allele specific oligonucleotide hybridisation can be used to identify any known mutation and involves differential hybridisation of sequence-specific oligonucleotides.
  • the oligonucleotides can be suitably prepared with the mutation placed centrally and are preferably hybridised to target DNA under conditions which permit hybridisation only if a perfect match is found.
  • oligonucleotides are hybridised to PCR-amplified target DNA (dot-blot technique), one mutation can be tested per reaction (Saiki et al Nature 324 163-166 (1986)), but when the allele specific oligonucleotides are attached to the hybridising membrane and hybridised with labelled target DNA (reverse dot-blot), a number of different mutations in one fragment can be tested (Saiki et al Proc. Natl. Acad Sci. USA 86 6230-6234 (1989)).
  • Microsatellite markers can be selected from available sources of genome information. Selection is preferably on the basis of the microsatellite motif (i.e. if there are differences), map location (such that they are evenly spaced throughout the genome) and the ability of their PCR primers to generate product.
  • Amplification of the corresponding target DNA sequences by PCR can be carried out conveniently using any available PCR protocol (McPherson eds. et al in "PCR: A
  • ASO probes can be achieved by standard chemical synthetic routes in the art comprising ligating together successive nucleotides and/or oligonucleotides.
  • ASO probes can also be prepared using reverse transcriptase to transcribe a desired RNA sequence in which the case the oligonucleotide will be a cDNA molecule.
  • Hybridisation of ASO probes to target DNA sequences may be performed using the dot-blot or reverse dot-blot techniques described above.
  • the ASO probe can be bound to a solid support which can be any suitable inert material, e.g. nitrocellulose, polyethylene, polypropylene, silicon (Lipshutz, R.J. et al,
  • a glass support with DNA probes bound to the surface can be termed a "DNA chip” or “oligonucleotide chip” and methods which use such embodiments are also within the scope of the present invention.
  • the production of DNA chips is comprehensively reviewed in Mirzabekov, A. in Trends Biotechnol. 12 27-32 (1994) and further described in
  • the ASO probe or probes bound to the surface of the inert support can be suitably arranged in the form of an array.
  • the several means by which an array can be made fall into three general categories: in situ (on-chip) synthesis of oligonucleotides or peptide nucleic acids (PNAs); arraying of prefabricated oligonucleotides/PNAs; and, spotting of DNA fragments.
  • the present invention is not limited with respect to the means chosen to prepare the array probes on a solid support.
  • the array can be prepared by photolithography or piezoelectric printing.
  • a mercury lamp is shone through a photolithographic mask onto the chip surface, which removes a photoactive group, resulting in a 5'-hydroxyl group capable of reacting with another nucleoside.
  • the mask can be used in this way to determine which nucleotides become activated. Successive rounds of deprotection and chemistry can result in oligonucleotides which are up to 30 bases in length.
  • the piezoelectric method uses a
  • printer-head (analogous to an ink-jet printer head) which travels across the array and at each spot to be applied, a microlitre drop of one of the four main bases (A, G,
  • oligonucleotide synthesis is carried out.
  • the method can be used to prepare oligonucleotides of up to 40-50 bases in length which represents an improvement over traditional controlled pore glass (CPG) oligonucleotide synthesis.
  • CPG controlled pore glass
  • arrays can also be simplified by prefabricating oligonucleotides or oligopeptides using conventional CPG methods and then by printing them onto the array using direct touch or micropipetting.
  • An alternative to this approach is to employ a controlled electric field to immobilise prefabricated oligonucleotides to spots (microelectrodes) on the array. This method uses biotinylated oligonucleotides which are directed to individual spots by polarising the charge at the spot and are then anchored at the spot via a streptavidin-containing permeation layer that covers the surface.
  • the third main method of preparing an array is to "spot" oligonucleotides directly onto the inert support surface.
  • glass slides can be over lay ed with a positively charged coating, such as amino silane or poly ly sine, and oligonucleotide fragments suspended in a denaturing solution are then printed directly onto the surface.
  • Analysis of the results of the hybridisation step can be achieved using standard techniques to detect the presence of chemical, fluorescent or radioactive labels attached to the target DNA sequence.
  • Chemical labels can include but are not limited to, biotin, avidin, horseradish peroxidase, alkaline phosphatase, or other visually detectable coloured dyes.
  • Fluorescent labels can include, but are not limited to, fluorescein, or other optically detectable fluorescent dyes.
  • Radioactive labels can include, but are not limited to, 32 P, 35 S, 14 C, 3 H, 125 I.
  • the analysis of the results can utilise laser desorbtion to interrogate the hybridisation of probe to target with a readout generated by mass spectrometry. Mass spectrometry can also be used alone or in conjunction with fluorescence or chemical markers depending upon the level of automation in the procedure.
  • a microsatellite repeat unit or Simple Sequence Repeat in the design of an allele specific oligonucleotide (ASO) probe.
  • an inert support comprising a plurality of allele specific oligonucleotide probes (ASOs) bound to the surface of the support, in which the ASOs specifically hybridise to a target DNA sequence which flanks a microsatellite repeat unit or Simple Sequence Repeat (SSR).
  • the support may any inert material as described above.
  • kits for the determination of an organisms genotype comprising an inert support in accordance with the fourth aspect of the invention and a means for detection the hybridisation of an ASO to a target DNA sequence as defined previously,
  • FIGURE 1 shows an example of maize allele specific oligonucleotides demonstrating hybridisation/non-hybridisation of the probes.
  • FIGURE 2 shows a comparison of sequences from Maize for microsatellite MACE01F07: Forward Primer.
  • FIGURE 3 shows a comparison of sequences from Septoria for 312F.
  • FIGURE 4 shows an alignment of pig SSR sequences for pig SH524.1 and pig SH525.1.
  • FIGURE 5 shows the conversion of microsatellite flanking sequences to ASOs and their use in genotyping 12 maize lines.
  • the 6 allele specific oligonucleotides (ASOs) and one control oligonucleotide designed from the flanking sequence are shown alongside the results of the genotyping presented as a compilation autoradiograph.
  • Marker assisted selection is already widespread in plant breeding.
  • the markers used in such plant breeding schemes include RFLPs, AFLPs and SSRs.
  • the main limitations of these gel-based technologies are that they both limit the number of samples that can be characterised to just a few hundred per day and increase the costs of the genetic screening.
  • the genetic material used in plant breeding is based upon extensive collections of semi-characterised lines, which, depending upon the species can vary from genetically identical and homozygous for all markers, to open pollinated material with a genetic structure similar to that found in animal populations.
  • Currently, a number of research groups are developing microsatellite markers for a range of crop species and these are rapidly becoming the marker of choice for high throughput genotyping.
  • Hybridisation/non-hybridisation of a probe to a selected DNA region can then be monitored via a suitable detection system.
  • This two state system hybridisation/non- hybridisation
  • This two state system is binary in nature as shown in Figure 1 and is ideal for interpretation by machines. If the single locus PCR product, hybridises to two allelic ASOs then the individual must be heterozygote, where as hybridisation to one ASO suggests that it is homozygote.
  • Line 1 TCTCTCTCTCTCTCTCGCTCTCTCTCGCGACGCTTGT7AACTCCTACT
  • Line 2 TCTCTCTCTCTCTCACTCTCTCTCTCCTCATATCACCCCCACT
  • Lines 7 and 8 are the same inbred line.
  • Figure 5 shows the results of genotyping 12 maize lines as a compilation autoradiograph with the ASOs shown in the Figure alongside their respective autoradiograph.
  • the ASOs were designed from the flanking sequences of the maize sequences shown in Figure 2.
  • the genotyping exercise successfully allowed determination of all the maize lines except for line T232.
  • by using the ASOs were designed from the flanking sequences of the maize sequences shown in Figure 2.
  • T232 is more closely related to T303, CO 159, B14, B37, B73 and F2 than it is to CM37, MO17, OH43, CO125 and F7.
  • Reverse primer 5 ' -CAAATATCTCTCATCTTTGCTGAC
  • Example 2 Example of variation in sequences flanking microsatellite repeat unit in Septoria The variation in the sequences flanking the microsatellite repeat units in Septoria, a fungal pathogen of wheat is as shown in Figure 3.
  • microsatellite primer sets have been previously characterised (Edwards, K.J. et al, BioTechniques 20 (5) 758-760 (1996)). Approximately five microsatellite markers for each of the available microsatellite motifs (CA, CT, CAA, etc) will be examined to evaluate the variability of the flanking sequences in the different SSR types. If there is a difference in the amount of variation in the flanking sequences, within the different motifs, then that specific type will be selected for future work. From the available microsatellites, 96 (mapped and characterised) markers will be selected for inclusion in this study. These will be selected on the basis of their microsatellites motif (if there are differences), map location (such that they are evenly spaced throughout the genome) and the ability of their PCR primers to generate a product from the chosen lines.
  • the 15 lines are: 1:A554, 2:B73, 3:CM37, 4:CO159, 5:F2, 6:H99, 7:MO17, 8:OH43, 9:PA91, 10:T232, ll:Tx303, 12:W64a, 13:CO125, 14:F7 and 15:F564.
  • Example 5
  • flanking primer sets for each of the 96 microsatellite markers will be used to amplify and sequence the corresponding loci from the 15 maize inbred lines.
  • a Biomek 2000 robot and a ABI377 automated sequencer will be utilised. Sequences generated will be compared via the programme CLUSTALW and the information generated used to design ASOs for the different genotypes. In the unlikely event of a marker not producing sufficient variation to design ASOs for each genotype then a linked marker will be sequenced in its place. ASOs for both the same locus and the different loci will be designed to have the same annealing temperatures (for an exact match).
  • the complexity of the ASO plate will be increased via the use of fluorescent dyes incorporated into the PCR products. Along with the use of suitable detection equipment, this should increase the number of loci per plate by at least a factor of four or it could be used to increase the number of individual plants screened per plate from one to four.
  • Another alternative approach will be to develop a silicon microchip version of the plate via photolithography based technology (Lipshutz et al., 1995).
  • Examples 8 to 10 Animals, specifically pigs Selective animal breeding is currently based on selection indices that take account of multiple selection objectives (i.e. selection for more than one trait at a time). Each trait of economic importance is likely to be influenced by several quantitative trait loci (QTL). To compete with traditional selective breeding practices, marker assisted selection will either have to deliver very large gains by focussing on major genes or else the ability to scan genomes for multiple QTL. To date marker assisted selection in animals has been restricted to a few major genes - for example, the 'Halothane' gene which causes porcine stress syndrome, ESR as a predictor of litter size in pigs, the mutation which causes bovine leukocyte adhesion deficiency GLAD. The current markers of choice for genome scanning in vertebrates (humans mice and farmed animals) are so-called microsatellites or simple sequence repeat (SSR) loci.
  • SSR simple sequence repeat
  • microsatellite loci There are more than a thousand microsatellite loci mapped in the pig. Information on most of these markers is freely accessible in the pig genome database (PiGBASE - http://www.ri.bbsrc.ac.uk/pigmap/pig_genome_mapping.html). Work will focus on evaluating the extent of sequence variation flanking microsatellite repeats and developing multiplex PCR for set of pig microsatellite markers.
  • Figure 4 shows an alignment of pig SSR sequences for pig SH524.1 and pig SH525.1 with differences marked by a " *" underneath the appropriate residue.
  • PCR amplification of dinucleotide repeats such as [dCdA]n which is the most abundant porcine microsatellite motif yields not only a fragment corresponding to the sequence as found in the template genomic DNA, but also minor artefactual products one and two repeat units smaller than the authentic product i.e. two and four nucleotides shorter.
  • the proportion of these 'stutter bands' relative to me authentic product varies from one microsatellite locus to the next. It has been demonstrated that these stutter or shadow bands arise solely from variation in the length of the dinucleotide repeats (Hauge, X.Y. and Lift, M., Nucl. Acids Res. 2, 411-415 (1993)).
  • the sequence of the DNA flanking the tandem repeats is faithfully reproduced during the PCR amplification.
  • Each microsatellite allele will be sequenced from both ends but only information from the primer to the start of the repeats will be gathered. Thus, the potential difficulties of interpreting sequence data from the authentic PCR product and the stutter bands which are out of register will be avoided.
  • the PCR products/alleles will be sequenced using cycle sequencing protocols and analysis on an ABI 373 DNA sequencer. Sequence analysis and comparison of the sequences from different alleles will be performed as described above for plants.
  • the current gel-based technology makes fewer demands for simultaneous genotyping of multiple markers as the gel analysis, which has a capacity for about 9 markers per individual per gel, is the rate limiting step. Therefore there have only been limited efforts to multiplex the PCR step of the genotyping of pig microsatellites - at present most markers are amplified independently and then the PCR products for up to 9 loci are pooled and analysed simultaneously on the ABI sequencer.
  • the DNA chip/ASO approach could require 96 or more markers to be analysed simultaneously for each individual.
  • the key number for automation in biology laboratories is 96 (8 x 12 matrices).
  • marker sets will be developed for multiplex PCR in groups of 8.
  • PCR optimisation for multiplexes will be conducted simultaneously with sequence analysis of marker sets optimised earlier.
  • the rate limiting step in the procedure will be the purification of the allelic PCR products from gels. If the requirement for gel purification can be restricted to agarose gels, the timetable for sequencing the allelic variation for the 96 selected markers can be condensed. Any saving in time arising in this manner will be used to sequence a larger sample of pigs in order to increase the probability of detecting more alleles. Alternatively, a comparison between DNA flanking microsatellite and other locations could be effected by analysis of randomly selected genomic sequences of similar size.
  • Non microsatellite loci would be isolated at random from small fragment genomic libraries, sequenced, PCR primers developed and the loci sequenced in all 20 pigs as outlined above. As the difficulties arising from the length variation and stutter bands inherent to microsatellite loci, these random control loci could be sequenced without recourse to gel purification and thus more quickly. These random loci would be assigned to chromosomes using the INRAière somatic hybrid cell panel (Robic, A. et al , Mammalian Genome 7, 438- 445 (1996)).

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Abstract

L'invention concerne une méthode de détermination du génotype d'un organisme, la méthode consistant à hybrider une sonde d'oligonucléotides spécifique à l'allèle (ASO) à une séquence d'ADN cible, la séquence d'ADN cible comprenant une région d'ADN flanquant une unité de répétition de microsatellite ou une répétition de séquence simple (SSR).
EP98932341A 1997-07-02 1998-07-02 Methode de determination du genotype d'un organisme par utilisation d'une sonde d'oligonucleotides specifique a l'allele s'hybridant a des regions flanquantes de microsatellites Withdrawn EP1002127A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
GB9714005 1997-07-02
GB9714005A GB9714005D0 (en) 1997-07-02 1997-07-02 Dna
GBGB9806702.8A GB9806702D0 (en) 1998-03-27 1998-03-27 Dna
GB9806702 1998-03-27
US8032598P 1998-04-01 1998-04-01
US80325P 1998-04-01
PCT/GB1998/001940 WO1999001576A1 (fr) 1997-07-02 1998-07-02 Methode de determination du genotype d'un organisme par utilisation d'une sonde d'oligonucleotides specifique a l'allele s'hybridant a des regions flanquantes de microsatellites

Publications (1)

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EP1002127A1 true EP1002127A1 (fr) 2000-05-24

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EP98932341A Withdrawn EP1002127A1 (fr) 1997-07-02 1998-07-02 Methode de determination du genotype d'un organisme par utilisation d'une sonde d'oligonucleotides specifique a l'allele s'hybridant a des regions flanquantes de microsatellites

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EP (1) EP1002127A1 (fr)
AU (1) AU8228098A (fr)
CA (1) CA2294037A1 (fr)
IL (1) IL133794A0 (fr)
WO (1) WO1999001576A1 (fr)

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CN108165614A (zh) * 2018-01-24 2018-06-15 安徽天勤农业科技有限公司 一种育种改进的玉米ssr标记快速银染检测方法

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WO2002020844A1 (fr) * 2000-09-08 2002-03-14 Polygenyx, Inc. Methodes et produits relatifs a l'analyse de genotypes a haut rendement
US6861034B1 (en) 2000-11-22 2005-03-01 Xerox Corporation Priming mechanisms for drop ejection devices
US6514704B2 (en) * 2001-02-01 2003-02-04 Xerox Corporation Quality control mechanism and process for a biofluid multi-ejector system
DE10237691B4 (de) * 2002-08-15 2010-01-28 Biotez Berlin-Buch Gmbh Biochemisch-Technologisches Zentrum Verfahren zum Nachweis von Einzelnukleotid-Polymorphismen (SNP) in Genen des Arzneimittelmetabolismus und Testkit zur Durchführung des Verfahrens
US20050260603A1 (en) 2002-12-31 2005-11-24 Mmi Genomics, Inc. Compositions for inferring bovine traits
CN107988406B (zh) * 2016-10-21 2019-08-20 江苏省中国科学院植物研究所 铁线莲耐热性基因位点qHR4的分子标记方法
CN107974509B (zh) * 2016-10-21 2019-08-20 江苏省中国科学院植物研究所 铁线莲耐热性基因位点qHR3的分子标记方法
CN107974508B (zh) * 2016-10-21 2019-08-20 江苏省中国科学院植物研究所 铁线莲耐热性基因位点qHR2的分子标记方法
CN107385044B (zh) * 2017-07-31 2020-10-27 中国疾病预防控制中心传染病预防控制所 球形孢子丝菌str分子标记及其应用
CA3230219A1 (fr) * 2021-09-10 2023-03-16 Michael Wigler Procede de mesure de variations de longueur de microsatellite

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US5633134A (en) * 1992-10-06 1997-05-27 Ig Laboratories, Inc. Method for simultaneously detecting multiple mutations in a DNA sample
WO1995015400A1 (fr) * 1993-12-03 1995-06-08 The Johns Hopkins University Genotypage par analyse simultanee de multiples loci a microsatellites
DE19525284A1 (de) * 1995-06-28 1997-01-02 Inst Pflanzengenetik & Kultur Mikrosatellitenmarker für Pflanzen der Spezies Triticum aestivum sowie des Tribus Triticeae und ihre Verwendung

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108165614A (zh) * 2018-01-24 2018-06-15 安徽天勤农业科技有限公司 一种育种改进的玉米ssr标记快速银染检测方法

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WO1999001576A1 (fr) 1999-01-14
AU8228098A (en) 1999-01-25
CA2294037A1 (fr) 1999-01-14
IL133794A0 (en) 2001-04-30

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