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WO1999025872A1 - Heterozygote screening process - Google Patents

Heterozygote screening process Download PDF

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Publication number
WO1999025872A1
WO1999025872A1 PCT/GB1998/003541 GB9803541W WO9925872A1 WO 1999025872 A1 WO1999025872 A1 WO 1999025872A1 GB 9803541 W GB9803541 W GB 9803541W WO 9925872 A1 WO9925872 A1 WO 9925872A1
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WO
WIPO (PCT)
Prior art keywords
sample
mutations
nucleic acid
gene
organism
Prior art date
Application number
PCT/GB1998/003541
Other languages
French (fr)
Inventor
Jamie William Foster
Alan John Schafer
Original Assignee
Hexagen Technology Limited
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 Hexagen Technology Limited filed Critical Hexagen Technology Limited
Priority to EP98955793A priority Critical patent/EP1032703A1/en
Priority to IL13622898A priority patent/IL136228A0/en
Priority to JP2000521235A priority patent/JP2002514382A/en
Priority to CA002311526A priority patent/CA2311526A1/en
Priority to AU12518/99A priority patent/AU1251899A/en
Publication of WO1999025872A1 publication Critical patent/WO1999025872A1/en

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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
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • 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

  • This invention relates to the screening of organisms carrying heterozygotic mutations.
  • the heterozygotic mutant population should satisfy two criteria. Firstly, mutations across the entire genome should be represented. This ensures that the population will contain .an individual carrying a mutation for any given gene. Secondly, for any given gene there should be more than one mutant-carrying individual. This ensures that a diverse range of mutations are available.
  • each individual can carry a large number of mutations and/or the population can be large.
  • a large number of mutations per individual is, however, undesirable for three principal reasons. Firstly, too many mutations per individual increases the likelihood of lethal consequences. Secondly, interactions between mutated genes can complicate matters. Thirdly, in order to draw valid conclusions about the effects of a mutation in a given gene it is necessary to ensure that mutations in other genes are not contributing towards the overall phenotype. This means that mutations in other genes must be removed by breeding and the burden of doing so increases with the number of mutations carried by an individual.
  • MBPs mismatch binding proteins
  • WO95/12689 suggests using immobilised MBPs for detecting heterozygosity in a sample from a single organism. This involves incubating amplified DNA with immobilised MBP and then investigating the bound heteroduplexes with labelled probe DNA [see also Wagner et al. (1995) Nucleic Acids Research 23:3944-3948].
  • nucleic acid which is denatured and re-annealed can hybridise to form heteroduplexes, whereas homozygous alleles (ie. neither sequence mutated) will form homoduplexes. Any heteroduplex will thus be made up of a normal strand and a mutated strand. If homoduplexes are removed (or, put another way, if heteroduplexes are enriched), the sample will represent those alleles in the original sample where one copy was a mutant form.
  • the final sample ie. the screening material
  • the final sample is enriched for heterozygous alleles.
  • two copies of a gene differ (eg. wild-type and mutant)
  • no heteroduplex will form.
  • a probe for a gene of interest may hybridise with a sample before homoduplex removal, therefore, it will only hybridise with the sample after homoduplex removal if the sample contained two different copies of the gene of interest.
  • a positive signal from the probe therefore indicates that the organism from which the sample was derived carried a mutant copy of the gene of interest.
  • the invention therefore provides a method for determining whether an organism carries a heterozygotic mutation in a gene of interest.
  • the sample is also suitable "genetic screening material" for use according to PCT/GB98/01945.
  • the nucleic acid sample will typically comprise DNA but may alternatively comprise RNA.
  • DNA is preferably in the form of cDNA, but genomic DNA may also be used.
  • Nucleic acid may be fragmented prior to the annealing or removal steps, preferably such that the fragments are 200-400bp in size. This can typically be achieved by using a restriction enzyme, although a plurality of restriction enzymes are preferably used to generate overlapping fragments.
  • the nucleic acid sample may be derived from various sources.
  • cDNA could be prepared from an individual's major organs and combined eg. to compensate for differential expression patterns.
  • the sample could be derived from a single organ or cell-type.
  • nucleic acid could be amplified [eg. see Abramson & Myers (1993) Curr Opin Biotech 4:41-47; Zh ⁇ ing et al. (1992) PNAS 89:5847-51]. If amplification is used, however, this preferably occurs after heteroduplex formation (ie. .after the s.ample is denatured and annealed) and after heteroduplex removal, due to the infidelity of polymerase replication used during .amplification. Any errors introduced in this way can result in the formation of a heteroduplex which could be misinte ⁇ reted as being due to the presence of a heterozygotic mutation, or which may reduce detection sensitivity by increasing background noise.
  • the amplification products are preferably treated to remove amplification errors (eg. see WO95/12689).
  • Stringent annealing conditions can be used to eliminate or reduce hybridisation between non-allelic loci.
  • mutant sequences can hybridise with normal sequences to form heteroduplexes although, of course, re-formation of homoduplexes can also occur between identical sequences (whether mutant or normal).
  • the degree of homoduplex removal should be sufficient so that the binding of a probe specific for a sequence of interest which was homozygous in the organism from which the sample is derived is quantitatively reduced. This will typically mean that the abundance of heteroduplexes relative to homoduplexes will be increased by at least two-fold, preferably by at least five-fold, and more preferably by at least ten-fold or greater (eg. 100-fold).
  • Homoduplexes may be removed using any reagent which binds to heteroduplexes in preference to homoduplexes (or vice versa) and which does not chemically modify or cleave its bound nucleic acid.
  • MBPs are preferred, such as MutS, along with its derivatives and homologues.
  • the MBP should be immobilised in some way (eg. WO95/12689).
  • the MBP could, for instance, be attached to a solid support. Suitable solid supports include cellulose, polystyrene, dextran, and nitrocellulose. These may be used in any suitable form, including beads (which may be magnetic), membranes, or columns.
  • the immobilised MBP is able to bind heteroduplexes and retain them on the solid support, whilst homoduplexes are not retained.
  • the bound nucleic acid may be eluted from the MBP [eg. Jolly et ⁇ l. (1997) Nucleic Acids Research 25:1913-1919], thus providing a sample which has been enriched for the presence of heteroduplexes ie. homoduplexes have been removed. If amplification is used, this may commence before or after elution. If amplification begins before elution, the amplification process will typically result in dissociation of the MBP/heteroduplex complex anyway (eg. during the denaturing step in PCR).
  • the nucleic acid sample may be derived from any diploid organism.
  • the organism is an animal, such as an insect, and preferably the animal is a vertebrate, such as a fish or a mammal.
  • Preferred mammals are rodents and hum-ans.
  • An organism '"carrying heterozygotic mutations can conveniently be prepared by mating two members of an in-bred (and therefore genetically homogenous) population, one of whose germ cells have been exposed to mutagenic conditions.
  • the gametes of this mutagenised organism will thus carry mutations, and the organism will transmit chromosomes carrying random mutations to its progeny.
  • the other organism, which has not been exposed to mutagen will transmit normal chromosomes.
  • the offspring of such a mating will receive one set of chromosomes from each parent and they will thus be heterozygous for every locus which was mutated in the mutagenised parent, because they will carry one chromosomes with a mutant allele and one chromosome with a normal allele (ie. heterozygous mutations). It is also possible to mutagenise both parent organisms, although this is less preferable. If a low level of mutation is induced, it is highly unlikely that an offspring will inherit two mut-ant copies (ie. homozygous mutations) of any given allele; mutations are still heterozygotic (ie. each allele has a mutant and normal copy), but mutations are present in both sets of chromosomes.
  • an organism is said to be "carrying heterozygotic mutations", therefore, it is meant (a) that the genome of the organism has been exposed to mutagenic conditions (b) the mutations which resulted from an exposure are only present in half of the diploid chromosomes (ie. heterozygotic - for every mutant allele, there is also a normal allele).
  • the frequency of heterozygotic mutations carried by the organism should thus be characteristic of exposure to mutagenic conditions, rather than reflecting spontaneous or background mutations, which are characterised by their low frequency of occurrence.
  • the heterozygous mutations should thus be present at a frequency substantially above this background frequency.
  • the mutation frequency is such that, on average, in every 50000 organisms or fewer (eg. one in every 10000 organisms, or one in every 1000 organisms) one mutant copy of a gene occurs which would cause .an altered phenotype if bred to homozygosity.
  • Many suitable methods for inducing mutations are known in the art. These include chemical mutagenesis, radiation, and retroviral or transposon insertion. Fuller accounts of the production of organisms carrying heterozygotic mutations can be found in W097/44485 and PCT/GB98/01945.
  • mutation refers to an alteration in the nucleotide sequence of a gene (including its regulatory sequences) from its wild-type or normal nucleotide sequence which results in the ability to form a heteroduplex with the corresponding normal gene.
  • the term does not include large scale deletions or chromosomal rearragements, but does include point mutations and small (l-4bp) deletions or insertions.
  • This includes mutations which may be phenotypically neutral, even when homozygous.
  • the range of these ""silent" mutations is diverse but, depending on the gene in question, they might be mutations in non-coding regions, point mutations which do not alter the function of a codon (eg. CCU to CCG, or CGG to AGG), and mutations which alter a codon but which ordinarily do not affect the final protein function, such as conservative amino acid substitutions (eg. CUU Leu to AUU He).
  • the invention also provides, therefore, a process for preparing a sample of screening material from an organism carrying heterozygous mutations, comprising the steps of:
  • the invention also provides a process for screening a population of organisms carrying heterozygous mutations in order to identify those members of the population which carry a mutation in a gene of interest, comprising the steps of:
  • the population comprises 100 or more organisms (eg. 1000, 10000, 100000, or more).
  • the samples of screening material are preferably arrayed prior to probing. This might take the form, for example, of fixing samples from 10000 individuals in a 100x100 array on a nitrocellulose membrane.
  • the probe is specific for the gene of interest and is labelled such that hybridisation between the probe and a sample of screening material can be detected.
  • This may be a radioactive label, for instance, or a fluorescent label.
  • the samples with which the probe hybridises are derived from an organism carrying a mutation in the gene of interest. It will, of course, be appreciated that whilst background or non-specific hybridisation might occur in s-amples which are not derived from an organism carrying a mutation in the gene of interest, this will be weaker than the hybridisation which occurs with a sample which is derived from an organism carrying a mutation in the gene of interest.
  • the samples of screening material from organisms not carrying a mutation in the gene of interest will, in fact, serve as controls for background or non-specific hybridisation, and hybridisation above the control level is indicative of a positive result.
  • hybridisation with a radioactive probe will occur at a low level throughout the array, but a number of samples will hybridise strongly with the probe.
  • the probing step can be adapted for the use of multiple probes. For instance, probes specific for different genes or specific for different regions in the same gene can be used simultaneously.
  • the invention also provides a process for screening a population of organisms carrying heterozygous mutations in order to identify those members of the population which carry mutations in a plurality of genes of interest.
  • probes are utilised, these are preferably differentially labelled (eg. differently coloured fluorochromes in each probe).
  • the population need not be screened at the level of the individual. For example, in a population of 10000 organisms, screening samples from groups of ten individuals could be combined to give 1000 combined screening samples. These are screened in the same way as the 10000 would be screened, but the probing results require deconvolution in order to determine which of the ten organisms in a positive combined screening sample carry a mutation. This would typically involve screening the ten separate samples which were originally pooled. Whilst combining samples in this way obviously reduces the initial screening effort (eg. from 10000 samples to 1000 samples), the sensitivity of the assay is reduced and the requirement for deconvolution arises. The choice of whether to combine and, if so, how many samples to combine (eg. 10, 50, 100 etc.) therefore depends on the available resources, the size of the population, the degree of homoduplex removal, etc. This choice can be made without difficulty by the sldlled person.
  • Figure 1 shows a scheme for generating mice carrying heterozygous mutations
  • Figure 2 shows how screening material from such mice can be prepared.
  • Figure 3 shows how a population of these mice can be screened to identify those members of the population which carry a mutation in the first exon of the Tyrp-1 gene.
  • Figure 4 shows the screening of the population for mutations in the first two exons of Tyrp-1 and in the first exon of cKit.
  • Figure 5 shows how pooling might be used according to the invention.
  • Random mutations were induced in the genome of premeiotic spermatogonia of 300 male mice (strain C3Heb/Fej) using ethylnitrosourea (ENU). Three separate doses of 100 mg/kg body weight ENU were injected interperitoneally, with each injection separated by a one week interval. Approximately one third of the mice were rendered permanently sterile, but after 8-14 weeks the other two thirds were mated with two non-mutagenised females each, producing FI offspring carrying heterozygous mutations in the genome of their somatic and germ tissue (see Figure 1). About 1000 FI offspring were generated per week in this way.
  • RNA and genomic DNA were extracted separately using stand.ard protocols (eg. Sambrook et al.). The RNA was converted to double stranded cDNA with reverse transcriptase.
  • DNA from each FI mouse was digested using Sau3Al in a total reaction volume of 30 ⁇ l (manufacturer's recommended conditions). Following digestion, the DNA was precipitated by the addition of 3 ⁇ l sodium acetate (3.0M) and 80 ⁇ l ethanol. The DNA was collected at the bottom of the reaction tubes [3] by centrifugation at 15,000 for 30 minutes .and the ethanol was aspirated and discarded.
  • the nucleic acid was denatured and re-annealed [4] using the phenol enhanced re-association technique (PERT) [Miller & Riblet (1995) Improved phenol emulsion DNA re-association technique (PERT) using thermal cycling.
  • PERT phenol enhanced re-association technique
  • the DNA was precipitated by adding 5 ⁇ l sodium acetate (3.0M) and 125 ⁇ l ethanol. The DNA was collected at the bottom of the reaction tubes by centrifugation at 15,000g for 30 minutes and the ethanol was aspirated and discarded. The DNA was re-suspended in 20 ⁇ l mismatch binding protein reaction buffer (20mM Tris-HCl, pH 7.6, 5mM MgCl 2 , O.lmM dithiothreitol, O.OlmM mM EDTA) and 3% BSA.
  • 20 ⁇ l mismatch binding protein reaction buffer (20mM Tris-HCl, pH 7.6, 5mM MgCl 2 , O.lmM dithiothreitol, O.OlmM mM EDTA
  • Each sample [5] was given an identifier so that the mouse from which any particular sample was derived could be traced, and the digested genomic DNA and cDNA samples were stored.
  • Disposable MBP columns [6] were prepared by applying 500ng MutS protein (Amersham UK) to nitrocellulose columns in 50 ⁇ l MBP reaction buffer. The DNA samples were removed from storage, applied to the columns and incubated at room temperature for 30 minutes to allow heteroduplex DNA molecules to be bound by the MutS MBP. Un-bound homoduplex molecules [9] were removed from the column by five washes [7] of 200 ⁇ l MBP reaction buffer ("Wash conditions").
  • Bound heteroduplex DNA molecules were eluted [8] by incubating the column at 37°C for 30 minutes in 50 ⁇ l elution buffer (lOOmM Tris-HCl, 150mM NaCl, lOOug/ml proteinase K). Eluted DNA [10] was precipitated by the addition of 5 ⁇ l sodium acetate (3.0M) and 125 ⁇ l ethanol and collected at the bottom of the reaction tube by centrifugation at 15,000g- for 30 minutes. The ethanol was aspirated and the DNA re- suspended in lO ⁇ l TE (lOmM Tris-HCl, pH 7.5, l.OmM EDTA).
  • This eluted heteroduplex DNA [10] can be used as screening material.
  • the amount of DNA in the blood samples was lower than that in the tail-clippings, thus reducing the quantity of heteroduplex DNA which was recovered from each sample.
  • the blood-derived samples of MBP-treated screening material were subjected to whole genome amplification [Zhang et al. (1992) supra] to give an unbiased linear amplification of the complete nucleic acid used as the starting material.
  • a 5 ⁇ l aliquot from each sample was placed in a reaction tube containing the whole genome amplification reaction components (lOOmM Tris-HCl, pH 8.9, 150mM KC1, 200 ⁇ M each [dCTP, dATP, dGTP, dTTP], 40mM random 15- mer, 1.25U Taq polymerase) and the samples were placed in a thermal cycling machine and cycled [94°C 1 minute; 37°C 2 minutes; 37°-55°C ramp at 10 seconds/degree; 55°C 4 minutes; 50 cycles], followed by a final incubation at 72°C for 5 minutes.
  • This amplified DNA can be used as screening material.
  • heteroduplex-enriched screening material was used in order to find F 1 mice which carried mutations in genes of interest. Because of the size of the population, it was anticipated that there would be more than one mouse carrying a mutation in any given gene, resulting in an allelic series of mutations spread throughout the coding and non-coding regions. In order to aid studies of the function of a gene, these mutations would preferably affect the function of the protein in different ways.
  • Tyrosine related protein- 1 (Tyrpl; Genbank accession number X03687), a melanocyte-specific enzyme involved in melanin synthesis. Recessive mutant alleles in mice cause a brown coat colour, while a dominant allele causes an almost white appearance in black mice.
  • Teyrpl Tyrosine related protein- 1
  • a probe [12] was produced by PCR amplification of the first exon of the gene from .an untreated C3Heb/Fej mouse, using a pair of specific primers which were designed using the PCR primer design program Primer-3.0. This DNA was radio-labelled using a multiprimeTM kit (Amersham, UK) inco ⁇ orating 32 P-dCTP, and hybridised [13] to the screening material on the nylon membranes.
  • the membranes were washed under stringent conditions [0.2xSSC, 0.2% SDS, 65°C] to reduce non-specific hybridisation and then they were exposed to X-ray film. Whilst a low level of radioactivity was visible across the whole film (background hybridisation), four dark spots were apparent on one of the autoradiograms, which were indicative of the probe being preferentially retained where there were complementary sequences. These spots were at positions A 16, C5, F10 and Jl 4 in the array, indicating that the mice which gave rise to these four samples carry heterozygotic mutations in the first exon of the Ty ⁇ l gene.
  • mice were selected from the population and are being subjected to phenotypic analysis. This will, of course, only identify phenotypes if the particular mutation is dominant. The mice are therefore being used for breeding to obtain mice carrying homozygous mutations for fuller functional characterisation. As the FI were heterozygous for mutations, only half of the F2 mice inherit the Ty ⁇ mutation from the parent and so, for the mutations to be earned homozygously, F3 progeny must be produced. Breeding to further generations (F4, F5, etc.) will also ensure that the contribution of any other mutations carried by the mice will be removed.
  • each of the mutations which were detected was determined by PCR amplification of the first exon of the Ty ⁇ l gene from the mouse's genome, followed by sequencing on an ABI automated sequencing machine. For each animal, at one position in the sequence there were two sequencing peaks, indicating that two different alleles were present. The four mutations were distributed across the first exon, giving an allelic series of mutations, albeit a small one.
  • MEF mast cell growth factor
  • SCF stem cell factor
  • MGF is the ligand for the c-kit tyrosine kinase receptor (in humans, c-KIT) and is a haematopoetic growth factor critical to growth of several distinct cell lineages. Mutations in MGF, or in c-kit, can affect the ligand/receptor interaction such that signalling is no longer triggered; mutations which have no such effect are also useful, since they can be used to map regions of the protein which are not critical for function.
  • the sites of hybridisation of the Ty ⁇ -1 probes were detected using ELISA inco ⁇ orating anti-biotin horse radish peroxidase (HRP).
  • the sites of hybridisation of the cKit probe were detected using ELISA inco ⁇ orating anti-fluorescein alkaline phosphatase (AP).
  • the spots which gave a positive HRP result revealed the identity of mice carrying heterozygous mutations within either the first or second exon of the Ty ⁇ -1 gene.
  • the same four mice as previously detected gave positive results, along with two further mice (K8 & M3). These mice were subsequently shown to carry heterozygous mutations in the second exon.
  • the spots which gave a positive AP result indicated by a blue colouration, were M5 and 014. Analysis of the mice from which these two samples were derived shows that they carried heterozygous mutations in the first exon of the c-kit gene.
  • FI mice In a further series of experiments, another 1500 FI mice were bred carrying heterozygotic mutations. At 6 weeks of age, however, these mice were sacrificed. Gametes (sperm or ova) and somatic tissue (spleen, kidney, heart and brain) were harvested from each FI mouse. cDNA was prepared from the somatic tissue, although genomic DNA could have been used instead. The cDNA from the different organs was combined to give a single combined cDNA sample for each FI mouse. The gametes were stored in labelled cryo-tubes in five 30x10 racks at -196°C and somatic tissue cDNA was stored in similar racks at -70°C. Each position in a gamete rack corresponded to a position in a somatic rack which contained material taken from the same mouse.
  • Gametes sperm or ova
  • somatic tissue spleen, kidney, heart and brain
  • the five somatic tissue cDNA racks were removed from storage, arrayed, and probed with the Ty ⁇ first exon probe in the same way as before. Two positive signals were seen, and the gametes corresponding to these somatic tissue samples were removed from the freezer. The gametes were used to produce F2 offspring for study as described above.

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Abstract

In order to facilitate the screening of an organism, or a population of organisms, carrying heterozygous mutations for identifying the presence of a mutation in a gene of interest, a process is provided which utilises mismatch binding proteins, such as MutS. The process comprises the steps of: obtaining a nucleic acid sample from the organism; denaturing any double stranded nucleic acid present in the sample; allowing the nucleic acid to anneal; and removing homoduplexes from the annealed sample. A positive signal from a probe specific for the gene of interest indicates that the organism carries a heterozygous mutation in said gene.

Description

HETEROZYGOTE SCREENING PROCESS
This invention relates to the screening of organisms carrying heterozygotic mutations.
Background to the invention
In order to study the effects of genetic mutation, it has been proposed to use .an in-bred population whose individuals carry heterozygotic mutations (eg. patent application WO97/44485; Schafer & Hawkins (1998) Nαtwre Biotechnology 16:33-39). This population can be screened at a genomic level (eg. using single-stend conformation polymorphism) in order to determine those individuals which carry a mutation in a given gene (ie. those which carry one mutant copy of a gene of interest and one normal copy), even though the effect of the mutation may not be manifested as a phenotype (eg. a recessive mutation). These individuals can then be bred to create organisms for functional studies which carry homozygotic mut-ant .alleles.
To be most useful, the heterozygotic mutant population should satisfy two criteria. Firstly, mutations across the entire genome should be represented. This ensures that the population will contain .an individual carrying a mutation for any given gene. Secondly, for any given gene there should be more than one mutant-carrying individual. This ensures that a diverse range of mutations are available.
These goals can be achieved in two ways: each individual can carry a large number of mutations and/or the population can be large.
A large number of mutations per individual is, however, undesirable for three principal reasons. Firstly, too many mutations per individual increases the likelihood of lethal consequences. Secondly, interactions between mutated genes can complicate matters. Thirdly, in order to draw valid conclusions about the effects of a mutation in a given gene it is necessary to ensure that mutations in other genes are not contributing towards the overall phenotype. This means that mutations in other genes must be removed by breeding and the burden of doing so increases with the number of mutations carried by an individual.
In order to reduce the number of mutations per individual whilst maintaining the overall number of mutations represented in a population, it is therefore necessary to increase population size. As population size incre.ases, however, the burden of mamtaining the population also increases (eg. a colony of 10,000 mice). Furthermore, the burden of screening the population also increases, in terms of both time and expense.
Whilst the former problem can be reduced by removing the need to maintain a living population whilst keeping the ability to screen the population and reproduce an individual of interest (eg. patent application PCT/GB98/01945, which uses two samples per individual: one for genetic screening and the other for reproductive purposes), the burden of screening a large population remains.
It is therefore an object of the invention to facilitate the screening of a population carrying heterozygous mutations in order to identify those members of the population which carry a mutation in a gene of interest.
Where two copies of a gene differ slightly in sequence (eg. point mutations), their nucleic acid str-ands can form heteroduplexes (ie. double stranded nucleic acid containing non-base-paired mismatches). Proteins are available which bind heteroduplexes in preference to homoduplexes and these are collectively known as "mismatch binding proteins" (MBPs) eg. bacterial protein MutS, which binds to mismatches in otherwise complementary nucleic acid duplexes.
WO95/12689 suggests using immobilised MBPs for detecting heterozygosity in a sample from a single organism. This involves incubating amplified DNA with immobilised MBP and then investigating the bound heteroduplexes with labelled probe DNA [see also Wagner et al. (1995) Nucleic Acids Research 23:3944-3948].
Description of the invention According to the invention, there is provided a process for preparing a sample of screening material from an organism carrying heterozygous mutations, comprising the steps of:
- obtaining a nucleic acid sample from the organism;
- denaturing any double stranded nucleic acid present in the sample;
- allowing the nucleic acid to anneal; and - removing homoduplexes from the annexed sample.
At heterozygotic positions in the genome (ie. one mut.ant allele and one normal allele), nucleic acid which is denatured and re-annealed can hybridise to form heteroduplexes, whereas homozygous alleles (ie. neither sequence mutated) will form homoduplexes. Any heteroduplex will thus be made up of a normal strand and a mutated strand. If homoduplexes are removed (or, put another way, if heteroduplexes are enriched), the sample will represent those alleles in the original sample where one copy was a mutant form.
If the organism carries one mutation in every 1000 genes, for instance, this has the effect of greatly reducing the complexity of each sample (eg. from 100,000 different sequences to 100 different sequences).
Furthermore, whereas the original sample represents both homozygous and heterozygous alleles, the final sample (ie. the screening material) is enriched for heterozygous alleles. Unless two copies of a gene differ (eg. wild-type and mutant), no heteroduplex will form. Whilst a probe for a gene of interest may hybridise with a sample before homoduplex removal, therefore, it will only hybridise with the sample after homoduplex removal if the sample contained two different copies of the gene of interest. A positive signal from the probe therefore indicates that the organism from which the sample was derived carried a mutant copy of the gene of interest. The invention therefore provides a method for determining whether an organism carries a heterozygotic mutation in a gene of interest.
The sample is also suitable "genetic screening material" for use according to PCT/GB98/01945.
The nucleic acid sample will typically comprise DNA but may alternatively comprise RNA. DNA is preferably in the form of cDNA, but genomic DNA may also be used. Nucleic acid may be fragmented prior to the annealing or removal steps, preferably such that the fragments are 200-400bp in size. This can typically be achieved by using a restriction enzyme, although a plurality of restriction enzymes are preferably used to generate overlapping fragments.
The nucleic acid sample may be derived from various sources. For instance, cDNA could be prepared from an individual's major organs and combined eg. to compensate for differential expression patterns. Alternatively, the sample could be derived from a single organ or cell-type.
In order to provide sufficient material for screening, rather than preparing nucleic acid from a large number of cells, nucleic acid could be amplified [eg. see Abramson & Myers (1993) Curr Opin Biotech 4:41-47; Zh∑ing et al. (1992) PNAS 89:5847-51]. If amplification is used, however, this preferably occurs after heteroduplex formation (ie. .after the s.ample is denatured and annealed) and after heteroduplex removal, due to the infidelity of polymerase replication used during .amplification. Any errors introduced in this way can result in the formation of a heteroduplex which could be misinteφreted as being due to the presence of a heterozygotic mutation, or which may reduce detection sensitivity by increasing background noise.
If amplification is used before heteroduplex formation, therefore, the amplification products are preferably treated to remove amplification errors (eg. see WO95/12689).
Stringent annealing conditions can be used to eliminate or reduce hybridisation between non-allelic loci. During .annealing, mutant sequences can hybridise with normal sequences to form heteroduplexes although, of course, re-formation of homoduplexes can also occur between identical sequences (whether mutant or normal).
The degree of homoduplex removal should be sufficient so that the binding of a probe specific for a sequence of interest which was homozygous in the organism from which the sample is derived is quantitatively reduced. This will typically mean that the abundance of heteroduplexes relative to homoduplexes will be increased by at least two-fold, preferably by at least five-fold, and more preferably by at least ten-fold or greater (eg. 100-fold).
Homoduplexes may be removed using any reagent which binds to heteroduplexes in preference to homoduplexes (or vice versa) and which does not chemically modify or cleave its bound nucleic acid. MBPs are preferred, such as MutS, along with its derivatives and homologues. The MBP should be immobilised in some way (eg. WO95/12689). The MBP could, for instance, be attached to a solid support. Suitable solid supports include cellulose, polystyrene, dextran, and nitrocellulose. These may be used in any suitable form, including beads (which may be magnetic), membranes, or columns. The immobilised MBP is able to bind heteroduplexes and retain them on the solid support, whilst homoduplexes are not retained. The bound nucleic acid may be eluted from the MBP [eg. Jolly et αl. (1997) Nucleic Acids Research 25:1913-1919], thus providing a sample which has been enriched for the presence of heteroduplexes ie. homoduplexes have been removed. If amplification is used, this may commence before or after elution. If amplification begins before elution, the amplification process will typically result in dissociation of the MBP/heteroduplex complex anyway (eg. during the denaturing step in PCR).
The nucleic acid sample may be derived from any diploid organism. Preferably the organism is an animal, such as an insect, and preferably the animal is a vertebrate, such as a fish or a mammal. Preferred mammals are rodents and hum-ans.
An organism '"carrying heterozygotic mutations" can conveniently be prepared by mating two members of an in-bred (and therefore genetically homogenous) population, one of whose germ cells have been exposed to mutagenic conditions. The gametes of this mutagenised organism will thus carry mutations, and the organism will transmit chromosomes carrying random mutations to its progeny. The other organism, which has not been exposed to mutagen, will transmit normal chromosomes. The offspring of such a mating will receive one set of chromosomes from each parent and they will thus be heterozygous for every locus which was mutated in the mutagenised parent, because they will carry one chromosomes with a mutant allele and one chromosome with a normal allele (ie. heterozygous mutations). It is also possible to mutagenise both parent organisms, although this is less preferable. If a low level of mutation is induced, it is highly unlikely that an offspring will inherit two mut-ant copies (ie. homozygous mutations) of any given allele; mutations are still heterozygotic (ie. each allele has a mutant and normal copy), but mutations are present in both sets of chromosomes.
Where an organism is said to be "carrying heterozygotic mutations", therefore, it is meant (a) that the genome of the organism has been exposed to mutagenic conditions (b) the mutations which resulted from an exposure are only present in half of the diploid chromosomes (ie. heterozygotic - for every mutant allele, there is also a normal allele).
It is thus apparent that an organism "carrying heterozygotic mutations" will typically not have been exposed to mutagenic conditions itself, but its genome (in the form of half of its chromosomes) will have been. The mutagenesis will have happened to an ancestor, but mutations induced in the genome of the ancestor's germ cells will be inherited by the organism "carrying heterozygotic mutations". This also ensures that any mutations detected in the screening material will be p.assed on by the organism, since the mutations are consistent throughout the org-anism's gametes and somatic tissue.
The frequency of heterozygotic mutations carried by the organism should thus be characteristic of exposure to mutagenic conditions, rather than reflecting spontaneous or background mutations, which are characterised by their low frequency of occurrence. The heterozygous mutations should thus be present at a frequency substantially above this background frequency. Suitably, the mutation frequency is such that, on average, in every 50000 organisms or fewer (eg. one in every 10000 organisms, or one in every 1000 organisms) one mutant copy of a gene occurs which would cause .an altered phenotype if bred to homozygosity. Many suitable methods for inducing mutations are known in the art. These include chemical mutagenesis, radiation, and retroviral or transposon insertion. Fuller accounts of the production of organisms carrying heterozygotic mutations can be found in W097/44485 and PCT/GB98/01945.
As used herein, "mutation" refers to an alteration in the nucleotide sequence of a gene (including its regulatory sequences) from its wild-type or normal nucleotide sequence which results in the ability to form a heteroduplex with the corresponding normal gene. Thus the term does not include large scale deletions or chromosomal rearragements, but does include point mutations and small (l-4bp) deletions or insertions. This, of course, includes mutations which may be phenotypically neutral, even when homozygous. The range of these ""silent" mutations is diverse but, depending on the gene in question, they might be mutations in non-coding regions, point mutations which do not alter the function of a codon (eg. CCU to CCG, or CGG to AGG), and mutations which alter a codon but which ordinarily do not affect the final protein function, such as conservative amino acid substitutions (eg. CUU Leu to AUU He).
The invention also provides, therefore, a process for preparing a sample of screening material from an organism carrying heterozygous mutations, comprising the steps of:
- obtaining two parent organisms;
- exposing the germ cells of one of the parent organisms to mutagenic conditions;
- mating the two parent organisms to produce an offspring organism carrying heterozygous mutations;
- obtaining a nucleic acid sample from the offspring organism;
- denaturing any double stranded nucleic acid present in the sample;
- allowing the nucleic acid to anneal; and
- removing homoduplexes from the annealed sample.
According to a further aspect of the invention, there is provided a sample of nucleic acid heteroduplexes obtainable by any of these processes.
The invention also provides a process for screening a population of organisms carrying heterozygous mutations in order to identify those members of the population which carry a mutation in a gene of interest, comprising the steps of:
- preparing samples of screening material for the members of the population, as described above; and - contacting the samples with a probe specific for said gene of interest.
Preferably the population comprises 100 or more organisms (eg. 1000, 10000, 100000, or more).
The samples of screening material are preferably arrayed prior to probing. This might take the form, for example, of fixing samples from 10000 individuals in a 100x100 array on a nitrocellulose membrane.
The probe is specific for the gene of interest and is labelled such that hybridisation between the probe and a sample of screening material can be detected. This may be a radioactive label, for instance, or a fluorescent label.
The samples with which the probe hybridises are derived from an organism carrying a mutation in the gene of interest. It will, of course, be appreciated that whilst background or non-specific hybridisation might occur in s-amples which are not derived from an organism carrying a mutation in the gene of interest, this will be weaker than the hybridisation which occurs with a sample which is derived from an organism carrying a mutation in the gene of interest. The samples of screening material from organisms not carrying a mutation in the gene of interest will, in fact, serve as controls for background or non-specific hybridisation, and hybridisation above the control level is indicative of a positive result.
In an array of 100x100 samples, for instance, hybridisation with a radioactive probe will occur at a low level throughout the array, but a number of samples will hybridise strongly with the probe.
It will be appreciated that the probing step can be adapted for the use of multiple probes. For instance, probes specific for different genes or specific for different regions in the same gene can be used simultaneously. Thus the invention also provides a process for screening a population of organisms carrying heterozygous mutations in order to identify those members of the population which carry mutations in a plurality of genes of interest.
Where multiple probes are utilised, these are preferably differentially labelled (eg. differently coloured fluorochromes in each probe).
It will also be appreciated that the population need not be screened at the level of the individual. For example, in a population of 10000 organisms, screening samples from groups of ten individuals could be combined to give 1000 combined screening samples. These are screened in the same way as the 10000 would be screened, but the probing results require deconvolution in order to determine which of the ten organisms in a positive combined screening sample carry a mutation. This would typically involve screening the ten separate samples which were originally pooled. Whilst combining samples in this way obviously reduces the initial screening effort (eg. from 10000 samples to 1000 samples), the sensitivity of the assay is reduced and the requirement for deconvolution arises. The choice of whether to combine and, if so, how many samples to combine (eg. 10, 50, 100 etc.) therefore depends on the available resources, the size of the population, the degree of homoduplex removal, etc. This choice can be made without difficulty by the sldlled person.
Brief description of the drawings Figure 1 shows a scheme for generating mice carrying heterozygous mutations, and Figure 2 shows how screening material from such mice can be prepared.
Figure 3 shows how a population of these mice can be screened to identify those members of the population which carry a mutation in the first exon of the Tyrp-1 gene. Similarly, Figure 4 shows the screening of the population for mutations in the first two exons of Tyrp-1 and in the first exon of cKit.
Figure 5 shows how pooling might be used according to the invention.
Examples
Generation of mice carrying heterozygotic mutations
Random mutations were induced in the genome of premeiotic spermatogonia of 300 male mice (strain C3Heb/Fej) using ethylnitrosourea (ENU). Three separate doses of 100 mg/kg body weight ENU were injected interperitoneally, with each injection separated by a one week interval. Approximately one third of the mice were rendered permanently sterile, but after 8-14 weeks the other two thirds were mated with two non-mutagenised females each, producing FI offspring carrying heterozygous mutations in the genome of their somatic and germ tissue (see Figure 1). About 1000 FI offspring were generated per week in this way.
Extraction of nucleic acid (Figure 2a)
At 6 weeks old, a 200μl blood sample and a short tail-clipping were taken from 3000 FI mice [1]. For each sample [2] from each mouse [1], RNA and genomic DNA were extracted separately using stand.ard protocols (eg. Sambrook et al.). The RNA was converted to double stranded cDNA with reverse transcriptase.
lOμg DNA from each FI mouse was digested using Sau3Al in a total reaction volume of 30μl (manufacturer's recommended conditions). Following digestion, the DNA was precipitated by the addition of 3μl sodium acetate (3.0M) and 80μl ethanol. The DNA was collected at the bottom of the reaction tubes [3] by centrifugation at 15,000 for 30 minutes .and the ethanol was aspirated and discarded. The nucleic acid was denatured and re-annealed [4] using the phenol enhanced re-association technique (PERT) [Miller & Riblet (1995) Improved phenol emulsion DNA re-association technique (PERT) using thermal cycling. Nucleic Acids Research 23:2339-2340; this technique is suitable for reducing hybridisation between non- allelic sequences]. The restriction digested, double-stranded DNA samples were re-suspended in 50μl PERT buffer (1.5M sodium thiocyanate, 120mM sodium phosphate, lOmM EDTA, 8% phenol) and denatured by heating to 100°C for 10 minutes and quickly chilled on ice to form an emulsion in the reaction. The samples were then placed in a programmable thermal cycler and cycled for 24 hours at 65°C (2 minutes) and 37°C (15 minutes). The samples were centrifuged at 15,000g for 10 minutes .and the upper phenol phase removed and discarded. The DNA was precipitated by adding 5μl sodium acetate (3.0M) and 125μl ethanol. The DNA was collected at the bottom of the reaction tubes by centrifugation at 15,000g for 30 minutes and the ethanol was aspirated and discarded. The DNA was re-suspended in 20μl mismatch binding protein reaction buffer (20mM Tris-HCl, pH 7.6, 5mM MgCl2, O.lmM dithiothreitol, O.OlmM mM EDTA) and 3% BSA.
Each sample [5] was given an identifier so that the mouse from which any particular sample was derived could be traced, and the digested genomic DNA and cDNA samples were stored.
Homoduplex removal (Figure 2b) Disposable MBP columns [6] were prepared by applying 500ng MutS protein (Amersham UK) to nitrocellulose columns in 50μl MBP reaction buffer. The DNA samples were removed from storage, applied to the columns and incubated at room temperature for 30 minutes to allow heteroduplex DNA molecules to be bound by the MutS MBP. Un-bound homoduplex molecules [9] were removed from the column by five washes [7] of 200μl MBP reaction buffer ("Wash conditions"). Bound heteroduplex DNA molecules were eluted [8] by incubating the column at 37°C for 30 minutes in 50μl elution buffer (lOOmM Tris-HCl, 150mM NaCl, lOOug/ml proteinase K). Eluted DNA [10] was precipitated by the addition of 5μl sodium acetate (3.0M) and 125μl ethanol and collected at the bottom of the reaction tube by centrifugation at 15,000g- for 30 minutes. The ethanol was aspirated and the DNA re- suspended in lOμl TE (lOmM Tris-HCl, pH 7.5, l.OmM EDTA).
This eluted heteroduplex DNA [10] can be used as screening material.
The amount of DNA in the blood samples was lower than that in the tail-clippings, thus reducing the quantity of heteroduplex DNA which was recovered from each sample. To compensate for this, the blood-derived samples of MBP-treated screening material were subjected to whole genome amplification [Zhang et al. (1992) supra] to give an unbiased linear amplification of the complete nucleic acid used as the starting material. A 5μl aliquot from each sample was placed in a reaction tube containing the whole genome amplification reaction components (lOOmM Tris-HCl, pH 8.9, 150mM KC1, 200μM each [dCTP, dATP, dGTP, dTTP], 40mM random 15- mer, 1.25U Taq polymerase) and the samples were placed in a thermal cycling machine and cycled [94°C 1 minute; 37°C 2 minutes; 37°-55°C ramp at 10 seconds/degree; 55°C 4 minutes; 50 cycles], followed by a final incubation at 72°C for 5 minutes.
This amplified DNA can be used as screening material.
Screening the population (Figure 3)
The samples of heteroduplex-enriched screening material were used in order to find F 1 mice which carried mutations in genes of interest. Because of the size of the population, it was anticipated that there would be more than one mouse carrying a mutation in any given gene, resulting in an allelic series of mutations spread throughout the coding and non-coding regions. In order to aid studies of the function of a gene, these mutations would preferably affect the function of the protein in different ways.
One gene of interest was the Tyrosine related protein- 1 (Tyrpl; Genbank accession number X03687), a melanocyte-specific enzyme involved in melanin synthesis. Recessive mutant alleles in mice cause a brown coat colour, while a dominant allele causes an almost white appearance in black mice. We were interested in studying mutations in the first exon of the tyrp gene [Figure 3 - 11]
lμl of each of the 3000 screening samples [10] was arrayed on nylon hybridisation membranes [13] in a grid pattern using a robotic fluid handling device (10 sheets; 300 samples per sheet: Al, A2, . . . , 019, 020). The nylon membranes were placed on absorbent paper soaked in 0.4M NaOH, simultaneously denaturing the screening samples and fixing them to the nylon membranes.
A probe [12] was produced by PCR amplification of the first exon of the gene from .an untreated C3Heb/Fej mouse, using a pair of specific primers which were designed using the PCR primer design program Primer-3.0. This DNA was radio-labelled using a multiprime™ kit (Amersham, UK) incoφorating 32P-dCTP, and hybridised [13] to the screening material on the nylon membranes.
The membranes were washed under stringent conditions [0.2xSSC, 0.2% SDS, 65°C] to reduce non-specific hybridisation and then they were exposed to X-ray film. Whilst a low level of radioactivity was visible across the whole film (background hybridisation), four dark spots were apparent on one of the autoradiograms, which were indicative of the probe being preferentially retained where there were complementary sequences. These spots were at positions A 16, C5, F10 and Jl 4 in the array, indicating that the mice which gave rise to these four samples carry heterozygotic mutations in the first exon of the Tyφl gene.
These four mice were selected from the population and are being subjected to phenotypic analysis. This will, of course, only identify phenotypes if the particular mutation is dominant. The mice are therefore being used for breeding to obtain mice carrying homozygous mutations for fuller functional characterisation. As the FI were heterozygous for mutations, only half of the F2 mice inherit the Tyφ mutation from the parent and so, for the mutations to be earned homozygously, F3 progeny must be produced. Breeding to further generations (F4, F5, etc.) will also ensure that the contribution of any other mutations carried by the mice will be removed.
In addition, the exact nature of each of the mutations which were detected was determined by PCR amplification of the first exon of the Tyφl gene from the mouse's genome, followed by sequencing on an ABI automated sequencing machine. For each animal, at one position in the sequence there were two sequencing peaks, indicating that two different alleles were present. The four mutations were distributed across the first exon, giving an allelic series of mutations, albeit a small one.
Creating an allelic series of mutations in a mouse using traditional transgenic methods would have been very labour intensive and time consuming. Screening the population of heterozygous mutant mice was far less onerous and gave satisfactory results. Simultøneouslv screening different genes (Figure 4)
We were also interested in studying mast cell growth factor (MGF in mice; known as stem cell factor (SCF) in humans).
MGF is the ligand for the c-kit tyrosine kinase receptor (in humans, c-KIT) and is a haematopoetic growth factor critical to growth of several distinct cell lineages. Mutations in MGF, or in c-kit, can affect the ligand/receptor interaction such that signalling is no longer triggered; mutations which have no such effect are also useful, since they can be used to map regions of the protein which are not critical for function. Dominant mutations of the MGF and c-kit genes in mice affect germ cell development, coat colour and hematopoiesis; mutation of human c-KIT can cause piebaldism, a pigmentation defect, and c-KIT mutations have also been found in mast cell leukemias.
Rather than simply repeating the Tyφ experiment with a MGF-specific probe, however, we developed the screening process so that we could identify mice carrying mutations in MGF at the same time as screening to find those carrying mutations in Tyφ.
This involved preparing a number of different probes. Three probes were used: (1) a probe [ 14] for the first exon of Tyφ (2) a probe [ 15] for the second exon of Tyφ (3) a probe [ 16] for the first exon of c-kit.
These probes were prepared as before, but the PCR primers used to prepare the Tyφ probes were labelled with biotin at their 5' ends and the primers used to prepare the cKit probe were labelled at their 5' ends with fluorescein. The labelled probes were mixed together and hybridised (stringent conditions) with another set of ten nylon membranes containing screening material.
The sites of hybridisation of the Tyφ-1 probes were detected using ELISA incoφorating anti-biotin horse radish peroxidase (HRP). The sites of hybridisation of the cKit probe were detected using ELISA incoφorating anti-fluorescein alkaline phosphatase (AP).
The spots which gave a positive HRP result, indicated by a red colouration, revealed the identity of mice carrying heterozygous mutations within either the first or second exon of the Tyφ-1 gene. The same four mice as previously detected gave positive results, along with two further mice (K8 & M3). These mice were subsequently shown to carry heterozygous mutations in the second exon. The spots which gave a positive AP result, indicated by a blue colouration, were M5 and 014. Analysis of the mice from which these two samples were derived shows that they carried heterozygous mutations in the first exon of the c-kit gene.
It will, of course, be appreciated that any suitable form of differentia labelling could have been used to distinguish between mutations in the different genes. The three pairs of PCR primers could, for instance, have been labelled with differently coloured fluorochromes. Furthermore, by combining this system with a pair of 32P labelled primers for a further gene, for example, four genes could be screened simultaneously etc.
Screening with pooling (Figure 5) Whilst the screening process used above offers significant advantages when compared with traditional methods, it still used ten nylon membranes per experiment. To reduce the effort required to screen the population, samples of screening material were pooled.
Prior to spotting on nylon membranes, samples of screening material were combined from groups often mice. This gave 300 combined samples, rather than 3000 individual s.amples.
These samples were probed as before with the 32P probe [12] for the first exon of the Tyφ gene. Four positive spots were detected [C8, G6, H3, 15]. To deconvolute the pooling, the ten samples which were combined to give sample C8 were then spotted individually on a small nylon membrane.
This was probed and one spot gave a positive signal. The mouse from which this sample was derived turned out to be mouse C5.
Paired samples
In a further series of experiments, another 1500 FI mice were bred carrying heterozygotic mutations. At 6 weeks of age, however, these mice were sacrificed. Gametes (sperm or ova) and somatic tissue (spleen, kidney, heart and brain) were harvested from each FI mouse. cDNA was prepared from the somatic tissue, although genomic DNA could have been used instead. The cDNA from the different organs was combined to give a single combined cDNA sample for each FI mouse. The gametes were stored in labelled cryo-tubes in five 30x10 racks at -196°C and somatic tissue cDNA was stored in similar racks at -70°C. Each position in a gamete rack corresponded to a position in a somatic rack which contained material taken from the same mouse.
The five somatic tissue cDNA racks were removed from storage, arrayed, and probed with the Tyφ first exon probe in the same way as before. Two positive signals were seen, and the gametes corresponding to these somatic tissue samples were removed from the freezer. The gametes were used to produce F2 offspring for study as described above.
It will, of course, be understood that the invention is described above by way of non-limiting example only and modifications may be made whilst remaining within the scope and spirit of the invention.

Claims

CLATMS
1. A process for preparing a sample of screening material from an organism carrying heterozygous mutations, comprising the steps of:
- obtaining a nucleic acid sample from the organism; - denaturing any double stranded nucleic acid present in the sample;
- allowing the nucleic acid to anneal; and
- removing homoduplexes from the annealed sample.
2. A process according to claim 1, wherein said organism is a mouse.
3. A process according to claim 1, further comprising the step of fragmenting the nucleic acid in the sample.
4. A process according to claim 1, wherein the removal of homoduplexes is effected using immobilised MutS protein.
5. A process according to claim 1, initially comprising the steps of:
- obtaining two parent organisms; - exposing the germ cells of one of the parent organisms to mutagenic conditions; and
- mating the two parent organisms to produce an offspring organism carrying heterozygous mutations;
6. A sample of nucleic acid heteroduplexes obtainable by a process according to any one of claims 1 to 5.
7. A process for screening a population of organisms carrying heterozygous mutations in order to identify those members of the population which carry a mutation in a gene of interest, comprising the steps of:
- preparing samples of screening material for the members of the population, according to claim 1 ; and - contacting the samples with a probe specific for said gene of interest.
8. A process according to claim 7, wherein said population comprises 100 or more organisms.
9. A process according to claim 7, wherein said samples are arrayed prior to probing.
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