WO2005028672A2 - Means and methods for mhc typing of cattle - Google Patents

Abstract

The present invention provides a method for identifying major histocompatibility complex (MHQ genes and/or allelic variants thereof in cattle comprising (i) providing a nucleic acid sample from a subject to be tested, (ii) amplifying a target region of the nucleic acid sample using a pair of nucleic acid primer molecules, the target region corresponding to an MIFIC gene or an identifying fragment thereof, (iii) exposing the amplification product(s) of step (ii) to a reference nucleic acid molecule corresponding to an MHC gene or a fragment thereof under conditions which permit hybridisation of complementary nucleic acid sequences, and (iv) measuring the mobility of nucleic acid heteroduplexes formed in step (iii) between the amplified target nucleic acid and the reference nucleic acid molecule, the mobility of the heteroduplexes being indicative of the MHC gene and/or allelic variant thereof present in the nucleic acid sample. Also provided are isolated polynucleotides and kits of parts for use in such methods.

Description

METHOD ANDPOLYNUCLEOTTDES THEREFOR

The present invention relates to means and methods for MHC typing of cattle. In particular, the invention relates to means and methods for determining the MHC I type of cattle using reference strand conformational analysis.

Background

Major histocompatibility complex (MHC) and tissue typing in humans

The major histocompatibility complex (MHC) represents one ofthe most polymorphic regions known. MHC class I and class II genes play a key role in the immune response, their high level of polymorphism enabling populations to survive alongside rapidly evolving pathogens (1). In humans, MHC genes are largely responsible for rejection of donated organs due to incompatibility between donor and recipient at one or more loci (2,3). Consequently 'tissue typing' systems in human have been constantly refined to allow better graft survival. Other reasons for tissue typing include to detect genes that are associated with disease, either to confirm the association or to predict the likelihood of a disease developing. Such associations relate primarily to autoimmune diseases, e.g. insulin dependent diabetes and ankylosing spondylitis (4). Large scale tissue typing of human populations has been performed for research purposes, for example to increase understanding of the evolution of the irnmune system. As DNA-based methods have become available, serological typing (5) has been replaced by sequence based PCR methods (6), which in turn have now been largely superseded by reference strand mediated conformation analysis (RSCA) (7,8).

MHC typing in domestic animals

There are two main reasons why it is important to develop an accurate, fast method of MHC typing in cattle.

(1) Some MHC haplotypes that may be common in Holstein Friesians (and other commercially important breeds) express a single class I gene (e.g. A18, A10). It is predicted that this would confer a significant disadvantage in immunological terms over a haplotype expressing 2 or 3 class I genes, because a much more restricted range of pathogen-derived peptides will be presented to T cells. At present, the incidence of single gene class I haplotypes, and MHC homozygotes, in cattle populations is unknown, as no met od is available to make this assessment. In addition, very limited data are available regarding the occurrence of any class I haplotype configurations. A further problem is that, at present, little is known about the functional capabilities of each of the 5 or 6 cattle class I genes. In humans, HLA-C appears to function primarily as a ligand for natural killer (NK) cells (14), but HLA-C is always accompanied by HLA-A and -B, which present antigen to T cells. It is possible that one or more of the cattle genes has a similarly specialised function, which may lead to problems due to variation in haplotypes (i.e. variable presence/absence). No information is currently available regarding polymorphism at each of the putative 5 or 6 loci. Current breeding strategies in large commercial farms, i.e. use of a relatively small number of different bulls, make it essential that populations / breeds are surveyed to generate information on haplotype and allelic diversity. If this is not carried out, breeding for other traits, e.g. meat / milk production, could inadvertently result in an immunologically compromised population. This is especially important with regard to disease control strategies, e.g. design and use of vaccines. It would also be useful to survey populations of rare / unusual breeds. These may become economically more important with increased public demand for organically produced and humanely produced meat and milk.

(2) There is evidence (in humans, mice and cattle) to suggest that pregnancy outcome / complications may depend in part at least on MHC compatibility between mother and foetus (15,16,17,18). Increased use in the cattle industry of artificial insemination and embryo transfer / freezing, and the trading of embryos across international boundaries, mean that it is important to be able to accurately determine the MHC class I type of semen / embryo donors and to have detailed information on the MHC genes present in recipient herds. There is also increasing commercial interest in cattle cloning, where MHC compatibility between mother and foetus may also be relevant.

MHC typing in domestic animals (cattle, pigs, sheep, horses) has previously relied largely on serological methods (9,10). Typing in these species has been performed primarily for research purposes, e.g. to increase understanding of the immune responses to infectious disease and to devise strategies to improve animal health. In humans, where three loci are expressed (HLA-A, B and C), it is possible to obtain a serological specificity for all three. In cattle, however, serological methods do not distinguish products of different class I loci, and consequently it was initially believed that cattle expressed a single class I gene. A similar problem has been encountered with the other domestic species. The reasons for this may relate to the very close proximity of the class I genes in such species compared to human class I genes (demonstrated in cattle and pig), and also that relatively few serological reagents are available (9). Recent publications have shown that cattle probably have 5 or 6 classical class I genes, with different haplotypes expressing between 1 and 3 of these (11), with none consistently expressed. Cattle appear to be unique in this respect. This introduces an additional level of complexity with regard to accurate MHC typing in cattle.

A PCR based typing system for use in cattle was devised in 1998 (12), but this method is only useful in a herd/population in which all of the

MHC class I genes have been previously identified and characterised, e.g. a closed, inbred herd. This PCR method, while useful, would not be applicable to large scale typing, unless a massive effort was put into sequencing all cattle MHC alleles. Although international bovine MHC (BoLA) workshops have been held (9,13) no large-scale assessment of

MHC class I diversity in cattle has ever been made, primarily due to lack of reagents and resources. Available data suggest that this could be as high as that recorded in humans (currently -800 alleles at 3 loci, EMBL-

EBI IMGT HLA database), due to the diversity of domestic cattle progenitor populations.

RSCA (Reference strand mediated conformation analysis)

Reference strand mediated conformation analysis (RSCA) is a conformational method, i.e. it does not depend on characterisation of nucleotide sequence, that offers high resolution and high sample throughput for MHC typing (or for analysis of other polymorphic genes). The MHC type is assigned based on accurate measurement of conformation-dependent DNA mobility in polyacrylamide gel electrophoresis. RSCA utilises fluorescent-labelled locus-specific reference DNAs to selectively modify the molecular conformation of the test DNAs. A full description of the method, and its advantages over other conformational methods, is given in Arguello & Madrigal, 1999 (8). In brief, the method utilises a locus-specific reference DNA fragment generated by PCR using locus-specific primers, one of which is labelled at the 5' end with a fluorescent dye. This reference strand is hybridised with PCR products from an unknown sample generated using the same locus-specific (but unlabelled) primers. Duplexes formed with the reference strand may be identified by a laser detection system after non-denaturing PAGE analysis, using an automated DNA sequencer {e.g. ABI PRISM 377), and GeneScan analysis software. Each allele gives rise to a heteroduplex of unique mobility, thus the number of heteroduplexes generated is equivalent to the number of alleles from that locus present in the sample.

Difficulties associated with using RSCA for MHC typing in cattle

The utility of RSCA methods for MHC typing was originally demonstrated in humans (8). More recently, the applicability of the RSCA method for MHC typing has also been demonstrated in sheep (22). However, the use of RSCA for MHC typing in the latter case was applied only to MHC class IT and was simplified greatly by the existence in sheep of only one expressed locus, and only one polymorphic exon. There are 21 full length cattle MHC class I cDNA sequences reported in the most recent BoLA workshop report (13), a number of wliich are not assigned to a particular haplotype / specificity. Subsequently, an additional 7 sequences from the Institute of Animal Health (IAH) laboratory have been approved by the BoLA Nomenclature Coirimittee and are available on EMBL/GenBank databases. The LAH laboratory has also obtained a further 10 full length sequences that have not been placed on public databases (Table 1). There are also large numbers of partial cattle MHC class I sequences on sequence databases. However, none of these is 'approved' by the Nomenclature Cornmittee, which has set certain conditions for acceptance of new alleles. These include the fact that full-length sequences are required, that sequencing should have been performed on both DNA strands, and that a minimum of 3 clones derived by PCR should be sequenced. Many of the partial sequences on the sequence databases are derived from genomic DNA. In these cases, it is impossible to know whether these are classical, non-classical, pseud^'ogenes or gene fragments. This means that these sequences are not useful in generating data for typing systems, which are only concerned with classical, expressed (i.e. functional) genes.

Thus, MHC genes in cattle exhibit a considerably greater degree of complexity than observed in either humans, with the existence of a putative 5 or 6 classical MHC class I loci in cattle being suggested in recent publications (11,19, Fig 1). The position of 3 of these loci within the MHC region of the genome is known (20). Sequence comparisons and phylogenetic analyses demonstrate inter-locus recombination, presumably caused by unequal crossing-over due to the very close proximity of the genes to one another. This means that identification of 'locus-specific' areas of sequence within the coding regions, which are required if RSCA are to be used, is problematic. This is less of a problem in human, where interlocus recombination does not occur, however locus-specific primers are still generally located in non-coding regions. The assignment of most cattle alleles to a locus is therefore speculative, and the BoLA Nomenclature Committee has thus far not included locus assignment (or naming of loci) in designation of new alleles.

Typing for class I genes does not necessarily require assignment of alleles to loci, but there are a number of reasons why it is strongly preferable. As aheady stated, the number and combination of class I genes expressed varies with haplotype (from one to three) in cattle. At present little information is available concerning the functional importance / polymorphism / expression levels of each of these genes. A typing system which simply generates data on the number of different alleles expressed by an individual would therefore be of little use with respect to the reasons outlined above for typing cattle. Because there is such limited full-length sequence data available, typing methods that rely on allele-specific sequence (e.g. PCR-SSP, 7) are not a viable option in cattle.

Hence, there exists a need to improved methods of MHC typing of other species, in particular cattle.

It is thus an object of the present invention to provide means and methods for MHC typing in cattle. Summary of Invention

According to the first aspect ofthe invention, there is provided a method for identifying a major histocompatibility complex (MHC) gene and/or allelic variants thereof in cattle comprising:

(i) providing a nucleic acid sample from a subject to be tested;

(ii) amplifying a target region ofthe nucleic acid sample using a pair of nucleic acid primer molecules, the target region corresponding to an MHC gene or an identifying fragment thereof;

(iii) exposing the amplification product(s) of step (ii) to a reference nucleic acid molecule corresponding to an MHC gene or a fragment thereof under conditions which permit hybridisation of complementary nucleic acid sequences; and (iv) measuring the mobility of nucleic acid heteroduplexes formed in step (iii) between the amplified target nucleic acid and the reference nucleic acid molecule

the mobility of the heteroduplexes being indicative of the MHC gene and/or allelic variant thereof present in the nucleic acid sample.

Thus, the invention provides a method for MHC typing of cattle which utilises reference strand mediated conformation analysis (RSCA).

By a 'major histocompatibility complex gene' we mean a member ofthe complex of genes coding for a large family of cell-surface proteins that bind peptide fragments of foreign proteins and present them to T lymphocytes to induce an immune response (for example, see Lewin et al, 1999, Immunol Rev. 167:145-58 and Amills et al., 1998, Rev Sci Tech. 17(1): 108-2). Preferably, the MHC gene is a Class I gene. By 'allelic variants' we mean naturally occurring variant forms of an MHC gene.

By 'cattle' we include domesticated bovine animals, i.e. animals of the Latin classification Bos taurus or Bos indicus. Preferably, the cattle are beef cattle and/or dairy cattle. More preferably, the cattle of selected from one ofthe following breeds: Hereford, Boran, Friesian and Angus.

In an alternative embodiment, the method of the first aspect of the invention is used to identify the MHC type of animals from rare breeds of cattle.

The term "nucleic acid sample" as used herein includes the meaning of any polynucleotide, typically RNA, cDNA or DNA, derivable directly or indirectly from the subject of interest. Preferably, the nucleic acid sample is a genomic DNA sample.

Samples for use in a method according to the first aspect ofthe invention may be in any form. When they are taken from the body of an animal, the sample is preferably ex vivo. This will typically be the case as body samples will be taken remotely and transferred to a testing facility for assay. The skilled person will appreciate that any type of sample may be used. Commonly, samples are epithelial lesions. Other suitable samples include blood samples, tissues, swabs and probangs (collection of fluid from the throat). Nucleic acid may be isolated from such samples by known methods (for example, see Sambrook and Russell, 2001, Molecular Cloning, A Laboratory Manual, (3rd Ed) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

By 'target region' in step (ii) we mean a region of the nucleic acid sample to be amplified, e.g. by PCR amplification, the target region corresponding to an MHC gene or an identifying fragment of an MHC gene. In a preferred embodiment, the target region corresponds to an MHC Class I gene or a fragment thereof. Thus, the target region amplified in step (ii) may correspond to an MHC Class 1 gene selected from the group consisting of group 1, group 2, group 3, group 4, group 5 and group 6 (formerly group la) genes, or a fragment thereof

By 'an identifying fragment' of an MHC gene we mean a fragment which is capable of being identified as belonging to a given class and/or allele of the MHC gene from which it is derived. In particular, the class and/or allele of the MHC gene fragment must be capable of being identified using the RSCA-based methods of the invention. MHC gene fragments may be tested for their suitability as identifying fragments using the methods ofthe invention.

Preferably, the target region amplified in step (ii) corresponds to a fragment of an MHC gene comprising exon 2, nitron 2 and exon 3.

In a preferred embodiment, step (ii) comprises PCR amplification of the target region.

Step (iii) of the method of the first aspect of the invention comprises exposing the amplification product(s) of step (ii) to a reference nucleic acid molecule corresponding to an MHC gene or a fragment thereof under conditions which permit hybridisation of complementary nucleic acid sequences.

The reference nucleic acid molecule is selected to correspond to the target region of the MHC region amplified in step (ii). Thus, for example, if the target region corresponds to a fragment of an MHC Class 1 gene comprising exon 2, intron 2 and exon 3, then the reference nucleic acid molecule corresponds to the same MHC Class 1 gene fragment. However, it will be appreciated by skilled persons that the reference nucleic acid molecule may correspond to an allelic variant which differs from that ofthe amplified target region. Indeed, any allelic variant ofthe MHC gene may serve as a reference nucleic acid molecule.

The choice of reference nucleic acid molecule, or 'reference strand', is important since use of different reference strands generates heteroduplexes with different mobilities. It is therefore advantageous to test a number of different reference strands, i.e. allelic variants, in order to choose the one that gives optimum resolution of alleles from a particular MHC locus.

Advantageously, the reference nucleic acid molecule is obtainable by PCR amplification of a nucleic acid sample from a subject of known MHC type using the same nucleic acid primer molecules of step (ii).

Advantageously, the following amplification primer molecules (e.g. PCR primers) are used in step (ii):

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting of the nucleotide sequence of SEQ ID NO:l (see Table 4) or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 1; and

(b) a second nucleotide acid molecule ('reverse primer') comprising or consisting of the nucleotide sequence of SEQ ID NO:2 (see Table 4) or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 2.

By 'a variant' polynucleotide we mean a polynucleotide in which one or more nucleotides is deleted, added or substituted relative to the defined sequence. Preferably, the variant shares at least 70% sequence identity with the specified nucleotide sequence, for example at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99 % sequence identity.

The percent sequence identity between two polynucleotides may be deteπnined using suitable computer programs, for example the GAP program ofthe University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polynucleotides whose sequence has been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows: Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM. By 'binding specifically' we mean a variant polynucleotide which, under high stringency conditions, displays detectable binding to the specified sequence, or complement thereof, whilst displaying substantially reduced, or preferably undetectable, binding to other polynucleotides having different sequences. Such conditions are well known in the art, for example see Sambrook and Russell (2001) Molecular Cloning, A Laboratory Manual, (3rd Ed) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

When the above forward and reverse primer sequences are used for amplification of the target sequence, step (iii) preferably comprises the use of a reference nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 3 or 4 (see Figure 5a and 5b, respectively), or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 3 or 4. More preferably, the reference nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 3 or 4.

In an alternative embodiment, the following amplification primer molecules are used in step (ii):

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting of or consisting of the nucleotide sequence of SEQ ID NO:5 (see Table 4) or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 5; and

(b) a second nucleotide acid molecule ('reverse primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 6 (see Table 4) or a variant thereof, wliich variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 6.

When the above forward and reverse primer sequences are used for amplification of the target sequence, step (iii) preferably comprises the use of a reference nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 7 (see Figure 5e), or a variant thereof, wliich variant is capable of binding specifically to the complementary sequence of SEQ ID NO: -7. More preferably, the reference nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 7.

In a further alternative embodiment, the following amplification primer molecules (e.g. PCR primers) are used in step (ii):

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting of the nucleotide sequence of SEQ LD NO: 8 (see Table 4) or a variant thereof, wliich variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 8; and

(b) a second nucleotide acid molecule ('reverse primer') comprising or consisting ofthe nucleotide sequence of SEQ ID NO: 9 (see Table 4) or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 9.

When the above forward and reverse primer sequences are used for amplification of the target sequence, step (iii) preferably comprises the use of a reference nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 10 or 11 (Figures 5c and 5d, respectively), or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 10 or 11. More preferably, the reference nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 10 or 11.

In a still further alternative embodiment, the following amplification primer molecules (e.g. PCR primers) are used in step (ii):

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting ofthe nucleotide sequence of SEQ ID NO: 12 or 14 (see Table 4) or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 12 or 14; and

(b) a second nucleotide acid molecule ('reverse primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 13 (see Table 4) or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 13.

When the above forward and reverse primer sequences are used for amplification of the target sequence, step (iii) preferably comprises the use of a reference nucleic acid molecule comprising or consisting of the nucleotide sequence of SEQ ID NO: 15 or 16 (Figures 5f and 5g, respectively), or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 3 or 4. More preferably, the reference nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 15 or 16.

In order to facilitate measurement of heteroduplexes, the reference nucleic acid molecule may comprise a detectable label. Any suitable labelling moiety known in the art may be used. In a preferred embodiment, the detectable label is capable of acting as a fluorescent marker, i.e. it capable of acting as a fluorescent marker if it can receive radiation at one wavelength (the excitatory wavelength) and, as a result, emit radiation at a different wavelength. Particularly preferred fluorescent markers include FAM (6-carboxy-fluorescein), TAMRA (6- carboxy-tetramefhyl-rhodamine), TET (tetrachloro-6-carboxy- fluorescein) and HEX (hexachloro-6-carboxy-fluorescein).

Conveniently, the detectable-labelled reference nucleic acid molecule is made by PCR amplification using a primer comprising a detectable, e.g. fluorescent, label.

In a preferred embodiment of the method of the first aspect of the invention, step (iii) comprises exposing the amplification product(s) of step (ii) to two or more distinct reference nucleic acid molecules, each reference nucleic acid molecule corresponding to at least part of a different allele of the same MHC gene. Such additional reference nucleic acid molecules are particularly useful for resolving ambiguous combinations of MHC gene allele present in the nucleic acid sample being tested. Thus, the amplification products of step (ii) may be separated into two or more aliquots, each aliquot being exposed to a different reference nucleic acid molecule and the mobility ofthe resultant heteroduplexes formed thereby measured. Conveniently, each reference nucleic acid molecule comprises an independently detectable label.

In step (iii), a reference nucleic acid molecule is annealed with amplification products of step (ii) under conditions which permit hybridisation of complementary nucleic acid sequences. By "conditions which permit hybridisation of complementary nucleic acid sequences" is included any conditions under which the reference nucleic acid molecule will display detectable binding, i.e. hybridisation, to amplification products of step (ii) having complementary sequences 5 whilst displaying substantially reduced, or preferably undetectable, binding to other polynucleotides having non-complementary sequences. Such hybridisation conditions, typically referred to as being of high stringency, are well known in the art, for example see Sambrook and Russell (2001) Molecular Cloning, A Laboratory Manual, (3rd Ed) Cold l o Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Advantageously, step (iii) comprises a first step of denaturing double- stranded nucleic acid molecules and a second step of allowing the resultant single-stranded nucleic molecules to re-anneal to each other. 15 for example, the double-stranded nucleic acid molecules may be denatured by heating at 95°C for 4 minutes and the resultant single- stranded nucleic molecules allowed to re-anneal by incubation at 55°C for 15 minutes followed by 15°C for 5 minutes. 0 It will be appreciated by skilled persons that step (iii) may lead to the formation of a number of different double-stranded nucleic acid species. For example, where the nucleic acid sample provided in step (i) is homozygous for a particular allele of an MHC gene, step (iii) may result in the formation of three double-stranded nucleic acid species; a 5 homoduplex ofthe amplified target region (i.e. the separated strands will re-anneal with themselves), a homoduplex of the reference nucleic acid strands, and a heteroduplex comprising one strand ofthe amplified target nucleic acid molecules produced in step (ii) and a second strand which is a reference nucleic acid molecule. Thus, by 'heteroduplex' we mean a 0 double-stranded nucleic acid molecule comprising one strand of amplified DNA, i.e. from amplification step (ii), and the other strand being a reference nucleic acid molecule. Where the reference nucleic acid molecule comprises a detectable label, only double stranded species comprising at least one such reference nucleic acid molecule will be detectable.

Step (iv) comprises measuring the mobility of nucleic acid heteroduplexes formed in step (iii). Preferably, heteroduplex mobility is measured by non-denaturing polyacrylamide gel electrophoresis (PAGE), for example as described in Sambrook & Russell, supra.

When using PAGE, it is important that the electrophoretic conditions are kept constant in order to minimise any effects on mobility of fluctuation in applied voltage, gel composition and temperature, etc. Such errors may be minimised further by the use of internal control markers (e.g. DNA size markers) and/or by taking measurements of mobility after the heteroduplex species have migrated the same distance.

Thus, in a preferred embodiment, a control marker is added to the nucleic acid heteroduplexes prior to separation by PAGE. More preferably, step (iv) comprises measuring the relative mobility of the nucleic acid heteroduplexes (compared to the mobility ofthe control marker).

Conveniently, step (iv) comprises detecting fluorescent-labelled nucleic acid heteroduplexes using a laser detection system. Such detection methods are well known in the art.

In a preferred embodiment of the method of the first aspect of the invention, the method further comprises step (v) of step (v) of sequencing one or more ofthe nucleic acid heteroduplexes. In a further preferred embodiment ofthe method ofthe first aspect ofthe invention, one or more of the steps is automated. By 'automated' we mean that one or more of the steps of the method are performed by a machine.

A second aspect of the invention provides an isolated polynucleotide comprising or consisting of the nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 5, 8, 12 and 14 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 1, 5, 8, 12 and 14, respectively.

By 'isolated' we mean that the polynucleotide is not located in a cell, i.e. in situ, but is suitable for in vitro use in the methods ofthe invention (see above).

Preferably, the isolated polynucleotide of the second aspect of the invention consists ofthe sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 5, 8, 12 and 14.

A third aspect of the invention provides an isolated polynucleotide comprising or consisting of the nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 6, 9, and 13 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 2, 6, 9, and 13, respectively.

Preferably, the polynucleotide consists of the sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 6, 9, and 13. A fourth aspect of the invention provides an isolated polynucleotide comprising or consisting of the nucleotide sequence selected from the group consisting of SEQ ID NOS: 3, 4, 7, 10, 11, 15 and 16 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 3, 4, 7, 10, 11, 15 and 16, respectively.

Preferably, the polynucleotide consists of the sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 3, 4, 7, 10, 11, 15 and 16.

A fifth aspect of the invention provides a pair of amplification primers comprising a first polynucleotide according to the second aspect of the invention and a second polynucleotide according to the third aspect of the invention.

Preferably, the primer pair is selected from the group consisting of the following primer pairs:

(a) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 2;

(b) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 5 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 6;

(c) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 8 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 9; (d) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 13; and

(e) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 14 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 12.

It will be appreciated by skilled persons that one or more of the polynucleotides ofthe invention may comprise a detectable label, such as a fluorescent label (see above).

Isolated polynucleotides of the second, third and fourth aspects of the invention can be produced by any suitable method. Conveniently they will be produced by synthetic methods well known in the art, usually utilising a solid-support such as controlled-pore glass or polystyrene (for a review see Sambrook and Russell (2001) Molecular Cloning, A Laboratory Manual, (3rd Ed) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and the references therein). Automated synthetic machines are available for this purpose and custom-synthesised polynucleotides of defined sequence can be obtained commercially from a number of companies (such as PE Applied Biosystems, Warrington, Cheshire, UK).

Isolated polynucleotides of the fourth aspect of the invention can also be produced by PCR. A sixth aspect of the invention provides the use of a polynucleotide of the second, third and/or fourth aspects of the invention for identifying major histocompatibility complex (MHC) genes and/or allelic variants thereof in cattle. 5 A seventh aspect ofthe invention provides a method of identifying major histocompatibility complex (MHC) genes and/or allelic variants thereof in cattle comprising:

l o (i) providing a nucleic acid sample from a subject to be tested; (ii) amplifying a target region ofthe nucleic acid sample using a pair of amplification primers according to the fifth aspect ofthe invention; and (iii) sequencing the amplification product(s) of step (ii). 15 Sequencing of the amplification product(s) of step (ii) may be accomplished using methods well known in the art (for example, see Sambrook & Russell, 2001, supra). 0 The skilled person will appreciate that there are many applications for the methods of the invention. The following examples are merely illustrative and non-limiting example ofthe potential uses ofthe methods ofthe invention:

25 (a) An eighth aspect ofthe invention provides a method for MHC typing of cattle comprising identifying one or more MHC genes or allelic variants thereof in a nucleic acid sample from a subject to be tested using a method according to the first aspect ofthe invention. By 'MHC typing' we mean determining the MHC genes, and/or allelic variants thereof, present in nucleic acid from the subject animal being tested.

(b) An ninth aspect of the invention provides a method of identifying the haplotype of an animal comprising identifying one or more MHC genes or allelic variants thereof in a nucleic acid sample from a subject to be tested using a method according to the first or seventh aspects ofthe invention.

By 'haplotype' we mean a set of closely linked MHC alleles (genes or DNA polymorphisms) inherited as a unit.

(c) An tenth aspect of the invention provides a method for selecting a pair of cattle for breeding comprising determining the MHC type of a first breeding pair member using a method according to the eighth aspect ofthe invention, and selecting a second breeding pair member having a MHC type compatible with the MHC type of a first breeding pair member.

By 'compatible' with respect to the MHC type of the breeding pair members we mean MHC types which lead to an increased likelihood of the progeny of the breeding pair members exhibiting a desired characteristic and/or a decreased likelihood of exhibiting an undesired characteristic. An example of an undesirable characteristic would be an animal that expresses two single gene class I haplotypes, such as A10 / A18. This would be less desirable than, for example, one single gene haplotype together with one that expressed two or three genes. (d) An eleventh aspect of the invention provides a method for selecting cattle for breeding comprising determining the MHC type of a population of candidate breeding pair members using a method according to the eighth aspect of the invention and selecting candidate breeding pair members for breeding having compatible MHC types.

(e) A twelfth aspect of the invention provides a method for selecting semen for use in artificial insemination in cattle comprising determining the MHC type of candidate semen using a method according to the eighth aspect of the invention and selecting semen for use in artificial insemination having a desired MHC type.

By 'desired MHC type' we include an MHC type which is compatible with the MHC type of the animal to be artificially inseminated with the selected semen.

(f) An thirteenth aspect ofthe invention provides a method for selecting a donor of semen for use in artificial insemination in cattle comprising determining the MHC type of a candidate semen donor using a method according to the eighth aspect of the invention and selecting a donor of semen for use in artificial insemination having a desired MHC type.

(g) An fourteenth aspect of the invention provides a method for selecting an embryo for use in artificial insemination in cattle comprising determining the MHC type of a candidate embryo using a method according to the eighth aspect of the invention and selecting a candidate embryo having a desired MHC type. Advantageously, the results ofthe methods ofthe invention are furnished in an intelligible format. Preferably, the results are recorded or stored on an information carrier. However, the step of furnishing the results could be by communicating the results orally.

By "information carrier", we include any means of storing information, such as paper, a computer disk; an internet-based information transfer system, such as an e-mail or internet page, or electronic file, etc. Of course, an "intelligible format" is also intended to embrace encrypted information which can be deciphered with an approximate key.

A fifteenth aspect of the invention provides a kit of parts for use in a method of the invention comprising a pair of amplification primers for use in a method of the invention. Preferably, the kit comprises an isolated polynucleotide according to the second aspect of the invention and an isolated polynucleotide according to the third aspect of the invention, or a primer pair according to the fifth aspect ofthe mvention.

In a preferred embodiment, the kit further comprises one or more reference nucleic acid molecules for use in a method ofthe invention, for example an isolated polynucleotide according to the fourth aspect of the invention.

Preferably, the kit comprises:

(a) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 1, an isolated polynucleotide comprising or consisting ofthe nucleotide sequence SEQ ID NO: 2 and an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 3 or 4; and/or

(b) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 5, an isolated polynucleotide comprising or consisting ofthe nucleotide sequence SEQ ID NO: 6 and an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 7; and/or

(c) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 8, an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 9 and an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 10 or 1; and/or

(d) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 12 or 14, an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 13 and an isolated polynucleotide comprising or consisting ofthe nucleotide sequence SEQ ID NO: 15 or 16.

It will be appreciated by persons skilled in the art that the kit of parts may additionally comprise one or more reagents for performing PCR and/or a control nucleic acid sample (such as a DNA ladder). Most preferably, the kit further comprises instructions for performing a method ofthe invention.

The invention will now be described in more detail by reference to the following Figures and Examples wherein:

Figure 1 shows a maximum likelihood tree of full-length cattle MHC class I sequences indicating putative 'groups' or loci.

Figure 2 is a schematic diagram of a bovine MHC class I gene from the promoter region to exon 4 (alpha 3 domain). Positions of generic and allele-specific primers used to amplify introns 1, 2 and 3 of the bovine alleles are shown (sequences are listed in Table 3).

Figure 3 shows the alignment of (a) partial intron 1 sequences, (b) partial intron 2 sequences and (c) partial intron 3 sequences from bovine MHC class I alleles of group 1 (dl8.4 and hdl), group 6 (formerly group la) (hd6 and man2), group 2 (dl8.3 and dl8.5), group 3 (18.2 and jspl) and group 4 (hd7 and dlδ.l). The alleles shown have been assigned to groups based on phylogenetic analysis of coding sequence. Potential locus specific sections of sequences have been highlighted.

Figure 4(a) shows the amplification of D18.4 and HD1 alleles (A14 and A31 haplotypes, respectively) using the group 1 specific primers, group 1 primer 4 (SEQ ID No. 1) and generic primer C (SEQ ID No. 2).

Lane 1 : phi x 174 ladder (DNA markers) Lane 2: A10/A10 Lane 3: Manus DNA Lane 4: Chicken DNA (negative control) Lane 5: A17/A17 Lane 6: A18/A18 Lane 7: A18/A17 Lane 8: A31/A31 (positive band observed) Lane 9: A13/A13 (positive band observed) Lane 10 A10/A20 Lane 11 All/All Lane 12 A10/A10 Lane 13 (empty lane)

Figure 4(b) shows the amplification of the group 6 alleles HD6, MAN2 and 3349.1 (A18, A19 and A17 haplotypes, respectively) using the group 6 specific primers, F 6c (SEQ ID No. 14 and R 6e (SEQ ID No. 13).

M: DNA markers Lane 1: A18/A18 (positive band observed) Lane 2: A19/A10 (positive band observed) Lane 3: A31/A31 Lane 4: A14/? Lane 5: A14/A14 Lane 6: All/All Lane 7: A17/A17 (positive band observed) Lane 8: A10/A10 Lane 9: A10/A20 Lane 10: (empty lane) M: DNA markers Figure 4(c) shows the amplification of group 2 alleles using the group 2 specific primers, F 2a (SEQ ID No. 5) and R 2c (SEQ ID No. 6). M: DNA markers Lane 1 : A 14/? (positive band observed) Lane 2: Al 1/A11 (positive band observed) Lane 3: A31/A31 Lane 4 : Al 9/A10 (positive band observed) Lane 5: A18/A18 Lane 6: All I All (positive band observed) Lane 7: A10/A10 (positive band observed) Lane 8: A10/A20 (positive band observed) Lane 9: (empty lane) M: DNA markers

Figure 4(d) shows the amplification of group 3 alleles using the group 3 specific primers, F 3c (SEQ ID No. 8) and R 3e (SEQ ID No. 9). M: DNA markers Lane 1 : Al 1/A11 (positive band observed) Lane 2: A10/A10 (positive band observed) Lane 3 : A14/? (positive band observed) Lane 4: A18/A18 Lane 5: A19/A10 (positive band observed) Lane 6: A31/A31 Lane 7: All I All Lane 8: A10/A20 (positive band observed) Lane 9: A14/A14 Lane 10: (empty lane) M: DNA markers

Figure 5 shows the sequences of the following exemplary reference strands:

(a) the D18.4 reference strand (SEQ ID No.3) for group 1 amplified by the group 1 specific primers, group 1 primer 4 (SEQ ID No. 1) and generic primer C (SEQ ID No. 2);

(b) the HDl reference strand for group 1 (SEQ LD No.4) amplified by the group 1 specific primers, group 1 primer 4 (SEQ ID No. 1) and generic primer C (SEQ ID No. 2);

(c) the A10 reference strand for group 3 (SEQ ID No. 10) amplified by the group 3 specific primers, F 3c (SEQ ID No. 8) and R 3e (SEQ ID No. 9); (d) the D18.2 reference strand for group 3 (SEQ ID No. 11) amplified by the group 3 specific primers, F 3c (SEQ ID No. 8) and R 3e (SEQ ID No. 9);

(e) the D18.3 reference strand for group 2 (SEQ ID No. 7) amplified by the group 2 specific primers, F 2a (SEQ ID No. 5) and R 2c (SEQ ID No. 6);

(f) the HD6 reference strand for group 6 (SEQ ID No. 15) amplified by the group 6 specific primers, F 6d (SEQ ID No. 12) and R 6e (SEQ LD No. 13); and

(g) the MAN2 reference strand for group 6 (SEQ ID No. 16) amplified the group 6 specific primers, F 6d (SEQ ID No. 12) and R 6e (SEQ ID No. 13).

Figure 6 shows RSCA data for Group I alleles, using either D18.4 as reference strand (panels a-c) or HDl as reference strand (panels d-f). Panels a and b both contain HDl, panel c contains an unknown Group 1 allele. Panels d and f both contain HDl, and panel e shows an unknown Group 1 allele.

Figure 7 shows RSCA data for Group 2 alleles, using either D18.3 as reference strand (panels a-c) or D18.5 as reference strand (panels d and e). Panels a and b both contain D18.5, panel c is an unknown Group 2 allele. Panels d and e both contain D 18.3.

Figure 8 shows RSCA data for Group 3 alleles, using either JSP.l as reference strand (panel a) or D18.2 as reference strand (panel b). Panel a contains unknown Group 3 alleles, panel b contains A10.

Figure 9 shows RSCA data for Group 6 alleles, using either HD6 as reference strand (panels, a, b) or MAN2 as reference strand (panel c). Panels a and b contain unknown Group 6 alleles. Panel c contains HD6.

EXAMPLES

Generation of group specific primers and reference strands

Sequence selection

Allele-specific primers for group 1, 2, 3, 4, and 6 alleles, as specified in Table 3, were used to generate the sequences of intron 1 (Fig 3 a), intron 2 (Fig 3b) and intron 3 (Fig 3c).

Sequence analysis revealed group-specific regions of sequence which were then used to generate a group-specific primers for PCR amplification (see Table 4). For example, the group 1-specific forward primer (Group 1 primer 4) when used in PCR with a generic reverse primer in exon 4 (Generic primer C) gave group 1-specific amplification, as demonstrated in Fig 4a (an amplification product is observed only in samples containing A13 and/or A31 haplotypes). The PCR product is 860 bp.

PCR amplification using PCR primers specific for group 6, group 2 and group 3 is shown in figures 4(b), (c) and (d), respectively. Thus, use of the group 6-specific primer pair, 6c and 6e (see Table 4), leads to amplification only in samples containing A17, A19 and A18 haplotypes (see figure 4b). Use of the group 2-specific primer pair, 2a and 2c (see Table 4), leads to amplification in samples containing A14 and Al l haplotypes (see figure 4c; amplification products are also observed in some other samples, indicating the presence of more than one gene on some A10 haplotypes and the presence of previously unidentified genes on A17 and A20 haplotypes). The use of the group 3-specific primer pair, 3 c and 3e (see Table 4), leads to amplification only in samples containing Al 1 and A10 haplotypes (see figure 4d).

The PCR conditions for amplification of group 1 alleles were: ~6ng DNA, 1 μM of each primer, 0.25mM dNTPs, 5μl lx PCR buffer, 1.5mM MgCl₂, 2.5 units Taq polymerase (to 50μl with water), with amplification cycles of: 98°C for 20 seconds (xl), followed by 96°C for 20 seconds, 68°C for 1.5 minutes (x35), 72°C for five minutes (xl). The conditions for amplification of group 2 and 3 alleles were 95°C for 5 minutes (xl), followed by 95°C for 30 seconds, 58.3°C for 50 seconds, 72°C for 1 minute (x35), 72°C for 5 minutes (xl). The conditions for amplification of group 6 alleles were the same except the annealing temperature is 59.8°C.

Amplification of alleles from each group (1, 2, 3 and 6) is shown in figure 4 (a to d).

These primers and conditions were subsequently used to generate group 1 reference strands from allele D18.4 (Figure 5a) and HD.l (Figure 5b), group 2 reference strands from alleles D18.3 (Figure 5e) and D18.5, group 3 reference strands from alleles JSP.l (A10) (Figure 5c) and D18.2 (Figure 5d), and group 6 reference strands from alleles HD6 (Figure 5f) and MAN2 (Figure 5g).

Reference strands can be generated for groups 4 and 5 using a similar strategy. MHC typing of cattle

Preparation of fluorescent-labelled reference strands (FLRs)

Group-specific primers were ordered from MWG Biotech. The forward primer in each case has a 5' TAMRA tag attached. These were made up to lOOμM then stored in aliquots of lOμM since repeated freeze thawing leads to degradation ofthe fluorescent label.

Alleles to act as reference were amplified from genomic DNA using unlabeled group-specific primers, cloned and sequenced. Minipreparations of DNA are stored at -20°C. Sequences were compared to Genbank sequences to ensure that they were the correct allele.

Fluorescent-labelled reference strands were prepared by amplifying the allele from the plasmids (containing the correct insert) using the labelled forward primer along with the unlabeled reverse primer. The PCR programme was the same as that used for amplification of test products. lμl of a 1/100 dilution of the miniprep DNA was used (diluted with TE or water). Samples were stored at -20°C prior to use.

Duplex formation

As detailed above, amplification of group 1 alleles from a test or unknown genomic DNA sample was performed with primers Group 1 primer 4 and Generic primer C (Table 4), using the same conditions as described above. Duplexes were formed by mixing 1 μl of FLR PCR product (200-3 OOng/μl) with 3μl of PCR product from the sample, and denatured at 95°C for 4 minutes to separate sense and antisense strands of DNA fragments present in the mixture. Reannealing was carried out at 55°C for 5 minutes followed by 15°C for 5 minutes. At this stage annealing occurs to form heteroduplexes, i.e. sense strand from the FLR product (labelled) annealed to antisense strands from the sample (unlabelled) PCR product, lμl of duplex solution was mixed with 0.5μl of internal length standard (GENESCAN-2500 Rox) to which 2μl of Ficoll loading buffer (6x) has been added.

Amplification of alleles of groups 2, 3, 4, 5 and 6 may be performed using the same methodology.

Electrophoresis and analysis of results

The duplexes were separated on a 48 cm, 0.2 n m-thick non-denaturing 6% polyacrylamide gel in lx TBE by electrophoresis on an ABI PRISM

377 sequencer at 1680v. Gel temperature was maintained at 40°C, and the running time was optimised for all groups at 12 hours.

Heteroduplexes formed with the fluorescent-labelled strand of the FLR and homoduplexes formed from the FLR were identified with the laser detection system of the sequencer, and were visualised as peaks with the

Genescan analysis software. If an individual is heterozygous for the gene being examined (and both alleles differ from the FLR), 3 peaks are observed (one corresponding to each allele, and the reference strand). At least 2 reference alleles should be tested for each gene in the first instance, to allow optimal separation.

As further cattle alleles are identified using this method, it may be necessary to test additional reference strands, in order to distinguish all alleles. It is possible to label different reference strands with different fluorescent markers, so that both FLRs can be used on the same gel and data analysed together.

Results

Figures 6 to 9 show RSCA data for each allele group.

Figure 6 (Group 1) shows 3 samples run using D18.4 as reference strand (panels a-c), and 3 samples run using HDl as reference strand (panels d- f). Panels a and b both contain HDl, panel c contains an unknown Group 1 allele. Panels d and f both contain HDl, and panel e shows an unknown Group 1 allele.

Figure 7 (Group 2) shows 3 samples run using D18.3 as reference strand (panels a-c) and 2 samples run using D18.5 as reference strand (panels d,e). Panels a and b both contain D18.5, panel c is an unknown Group 2 allele. Panels d and e both contain D18.3.

Figure 8 (Group 3) shows 1 sample run using JSP.l as reference strand (panel a) and one using D18.2 as reference strand (panel b). Panel a contains unknown Group 3 alleles, panel b contains A10.

Figure 9 (Group 6) shows 2 samples run using HD6 as reference strand (panels, a, b) and one sample run using MAN2 as reference strand (panel c). Panels a and b contain unknown Group 6 alleles. Panel c contains HD6.

These data demonstrate use of the method of the first aspect of the invention. For example, they show that the same allele amplified from different animals (e.g. Figure 6, panels a and b) shows the same mobility. Table 1

Allele Ref/Accession no Haplotype Breed Group

BL3-6 M21044 Aw33 Hereford 3

BL3-7 M21043 Aw33 Hereford 2

A10(5.1) M69206 A10 Boran 3

JSP.l X92870 A10 Friesian 3

JSP.2 Unpublished A10 Boran 3

JSP.3 Unpublished A10 Boran 3

KN104 M69204 KN104 Boran 5

BSA L02832 A2. A30 Angus 3

BSF L02834 A2, A30 Angus 2

BSN L02835 A2, A30 Angus 2

BSX U01187 A2, A30 Angus 1

HD6 X80934 A18 Friesian 6

HDl X80933 A31 Friesian 1

HD7 X80935 A31 Friesian 2

D18.1 Y09205 A14 Friesian 4

D18.2 Y09206 Al l Friesian 3

D18.3 Y09207 All Friesian 2

D18.4 Y09208 A14 Friesian 1

MAN2 AJ010861 A19 Angus 6

3349.1 AJ010862 A17 Friesian 6

MANl AJ010863 A12 Angus 2

MAN3 AJ010864 A12 Angus 1

4222.1 AJ010865 A12 Friesian 1

MAN8 AJ010866 A19 Angus 2

E55.1 AY188804 A25 Boran? 1

E55.2 AY188805 A25 Boran? 2

E223.1 AY188802 KN12 Boran 1

E223.2 AY188803 KN12 Boran 4

F187.1 AY188799 KN103 Boran 3

E54.1 AY188801 KN8 Boran 3

Table 1 lists representative full-length cattle MHC class I sequences used in the study. Database accession number (GenBank/EMBL) and / or reference are shown. 'Haplotype' refers to serologically defined haplotype/specificity in which the allele is expressed, if known. 'Group' refers to putative locus/group (see Fig 1). Table 2

Locus / group Allele Haplotype Map position 1 HDl A31 D18.4 A14 Yes MAN3 A30

2 D18.5 A14 Yes D18.3 Al l MAN8 A19

3 JSP.l AlO D18.2 All 5.1 A10*

4 D18.1 A14 Yes

5 KN104 KN104

6 HD6 A18 MAN2 A19 3349.1 A17

Table 2 lists the alleles used for design of locus-specific primers. 'Haplotype' refers to the serologically determined haplotype in which the allele is expressed, and 'map position' refers to presence of gene in A14 homozygous BAC library.

Table 3

Sequences of primers used to amplify introns 1, 2 and 3 from 10 bovine MHC class I alleles

(a) Intron 1 (F= forward primer. R= reverse primer)

TABLE 3 - continued

Co ) Intron 2 (F= forward primer, R= reverse primer)

TABLE 3 - continued

(c) Intron 3 (F ^forward primer R= reverse primer, all alleles amplified with reverse intron3 generic primer unless otherwise stated)

Table 3 lists the primers used to amplify introns 1 (Table 3 a), 2 (Table 3b) and 3 (Table 3c). 10 In most cases PCR conditions were optimised for melting temperature for each primer pair to ensure specific amplification.

15

Table 4 Sequence of group specific primers

*R=AorG, **Y=CorT

References

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Claims

1. A method for identifying major histocompatibility complex (MHC) genes and/or allelic variants thereof in cattle comprising:

(i) providing a nucleic acid sample from a subject to be tested; (ii) amplifying a target region of the nucleic acid sample using a pair of nucleic acid primer molecules, the target region corresponding to an MHC gene or an identifying fragment thereof; (iii) exposing the amplification product(s) of step (ii) to a reference nucleic acid molecule corresponding to an MHC gene or a fragment thereof under conditions which permit hybridisation of complementary nucleic acid sequences; and (iv) measuring the mobility of nucleic acid heteroduplexes formed in step (iii) between the amplified target nucleic acid and the reference nucleic acid molecule the mobility ofthe heteroduplexes being indicative of the MHC gene and/or allelic variant thereof present in the nucleic acid sample.

2. A method according to Claim 1 wherein the nucleic acid sample provided in step (i) is genomic DNA.

3. A method according to Claim 1 or 2 wherein the target region amplified in step (ii) corresponds to an MHC Class I gene or a fragment thereof.

4. A method according to any one of the preceding claims wherein the target region amplified in step (ii) corresponds to an MHC Class I gene selected from the group consisting of group 1, group 2, group 3, group 4, group 5 and group 6 genes, or a fragment thereof

5. A method according to Claim 3 or 4 wherein the target region amplified in step (ii) corresponds to a fragment of an MHC gene comprising exon 2, intron 2 and exon 3.

6. A method according to any one of the preceding claims wherein step (ii) comprises PCR amplification.

7. A method according to any one of the preceding claims wherein, in step (ii), the following amplification primer molecules are used:

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 1; and (b) a second nucleotide acid molecule ('reverse primer') comprising or consisting ofthe nucleotide sequence of SEQ ID NO: 2 or a variant thereof, which variant is capable of binding • specifically to the complementary sequence of SEQ ID NO: 2

8. A method according to Claim 7 wherein the forward primer comprises or consists of the nucleotide sequence of SEQ ID NO: 1 and the reverse primer comprises or consists of the nucleotide sequence of SEQ ID NO: 2.

9. A method according to Claim 7 or 8 wherein the reference nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 3 or 4, or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ LD NO: 3 or 4.

10. A method according to Claim 9 wherein the reference nucleic acid molecule consists ofthe nucleotide sequence of SEQ ID NO: 3 or 4.

11. A method according to any one of Claims 1 to 6 wherein, in step (ii), the following amplification primer molecules are used: (a) a first nucleotide acid molecule ('forward primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 5 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 5; and (b) a second nucleotide acid molecule ('reverse primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 6 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 6

12. A method according to Claim 11 wherein the forward primer comprises or consists of the nucleotide sequence of SEQ ID NO: 5 and the reverse primer comprises or consists of the nucleotide sequence of SEQ ID NO: 6.

13. A method according to Claim 11 or 12 wherein the reference nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 7 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 7.

14. A method according to Claim 13 wherein the reference nucleic acid molecule consists ofthe nucleotide sequence of SEQ ID NO: 7.

15. A method according to any one of Claims 1 to 6 wherein, in step (ii), the following amplification primer molecules are used:

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 8 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 8; and (b) a second nucleotide acid molecule ('reverse primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 9 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 9

16. A method according to Claim 15 wherein the forward primer comprises or consists of the nucleotide sequence of SEQ ID NO: 8 and the reverse primer comprises or consists of the nucleotide . sequence of SEQ ID NO: 9.

17. A method according to Claim 15 or 16 wherein the reference nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 10 or 11, or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 10 or 11.

18. A method according to Claim 19 wherein the reference nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 10 or 11.

19. A method according to any one of Claims 1 to 6 wherein, in step (ii), the following amplification primer molecules are used:

(a) a first nucleotide acid molecule ('forward primer') comprising or consisting of the nucleotide sequence of SEQ ID NO: 12 or 14, or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 12 or 14; and (b) a second nucleotide acid molecule ('reverse primer') comprising or consisting ofthe nucleotide sequence of SEQ ID NO: 13 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 13.

20. A method according to Claim 19 wherein the forward primer comprises or consists of the nucleotide sequence of SEQ ID NO: 12 or 14 and the reverse primer comprises or consists of the nucleotide sequence of SEQ ID NO: 13.

21. A method according to Claim 19 or 20 wherein the reference nucleic acid molecule comprises or consists of the nucleotide sequence of SEQ ID NO: 15 or 16, or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 15 or 16.

22. A method according to Claim 21 wherein the reference nucleic acid molecule consists of the nucleotide sequence of SEQ ID NO: 15 or 16.

23. A method according to any one of the preceding claims wherein the reference nucleic acid molecule comprises a detectable label.

24. A method according to Claim 23 wherein the detectable label is fluorescent.

25. A method according to any one of the preceding claims wherein the reference nucleic acid molecule is obtainable by PCR amplification of a nucleic acid sample from a subject of known MHC type using the nucleic acid primer molecules of step (ii).

26. A method according to any one ofthe preceding claims wherein step (iii) comprises exposing the amplification product(s) of step (ii) to two or more distinct reference nucleic acid molecules, each reference nucleic acid molecule corresponding to at least part of a different MHC gene allele.

27. A method according to Claim 26 wherein each reference nucleic acid molecule comprises an independently detectable label.

28. A method according to any one of the preceding claims wherein step (iii) comprises a first step of denaturing double-sfranded nucleic acid molecules and a second step of allowing the resultant single- stranded nucleic molecules to re-anneal to each other.

29. A method according to Claim 28 wherein the double-stranded nucleic acid molecules are denatured by heating at 95°C for 4 minutes and the resultant single-stranded nucleic molecules are re- annealing by incubation at 55°C for 15 minutes followed by 15°C for 5 minutes.

30. A method according to any one of the preceding claims wherein step (iv) comprises measuring the mobility of nucleic acid heteroduplexes by non-denaturing polyacrylamide gel electrophoresis (PAGE).

31. A method according to Claim 30 wherein the mobility measurements are taken after the heteroduplex species have all moved a predetermined distance.

32. A method according to Claim 31 wherein step (iv) comprises measuring the mobility ofthe nucleic acid heteroduplexes relative to the mobility ofthe control nucleic acid species.

33. A method according to any one of the preceding claims wherein step (iv) comprises detecting fluorescent-labelled nucleic acid heteroduplexes using a laser detection system.

34. A method according to any one of the preceding claims further comprising step (v) of sequencing one or more of the nucleic acid heteroduplexes.

35. A method according to any one of the preceding claims wherein one or more ofthe steps therein is automated.

36. An isolated polynucleotide comprising or consisting ofthe nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 5, 8, 12 and 14 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 1, 5, 8, 12 and 14, respectively.

37. An isolated polynucleotide according to Claim 36 wherein the polynucleotide consists of the sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 5, 8, 12 and 14.

38. An isolated polynucleotide comprising or consisting ofthe nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 6, 9, and 13 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 2, 6, 9, and 13, respectively.

39. An isolated polynucleotide according to Claim 38 wherein the polynucleotide consists of the sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 2, 6, 9, and 13.

40. An isolated polynucleotide comprising or consisting ofthe nucleotide sequence selected from the group consisting of SEQ ID NOS: 3, 4, 7, 10, 11, 15 and 16 or a variant thereof, which variant is capable of binding specifically to the complementary sequence of SEQ ID NO: 3, 4, 7, 10, 11, 15 and 16, respectively.

41. An isolated polynucleotide according to Claim 40 wherein the polynucleotide consists of the sequence of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 3, 4, 7, 10, 11, 15 and 16.

42. An isolated polynucleotide according to Claim 36 or 41 comprising a detectable label.

43. An isolated polynucleotide according to Claim 42 wherein the label is a fluorescent label.

44. A pair of amplification primers comprising an isolated polynucleotide according to Claim 36 or 37 and an isolated polynucleotide according to Claim 38 or 39.

45. A pair of amplification primers according to Claim 44 wherein the primer pair is selected from the group consisting of the following primer pairs:

(a) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 2; (b) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 5 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 6; (c) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 8 and a reverse primer which comprises or consists ofthe nucleotide sequence of SEQ ID NO: 9; (d) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 12 and a reverse primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 13; and (e) a forward primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 14 and a reverse primer which comprises or consists of the nucleotide sequence of SEQ ID NO: 12.

46. Use of a polynucleotide as defined in any one of Claims 36 to 43 for identifying major a histocompatibility complex (MHC) gene and/or an allelic variant thereof in cattle.

47. A method of identifying major histocompatibility complex (MHC) genes and/or allelic variants thereof in cattle comprising:

(i) providing a nucleic acid sample from a subject to be tested; (ii) amplifying a target region of the nucleic acid sample using a pair of amplification primers according to Claim 44 or 45; and (iii) sequencing the amplification product(s) of step (ii).

48. A method for MHC typing of cattle comprising identifying one or more MHC genes or allelic variants thereof in a nucleic acid sample from a subject to be tested using a method according to any one of Claims 1 to 35 or 47.

49. A method of identifying the haplotype of an animal comprising identifying one or more MHC genes or allelic variants thereof in a nucleic acid sample from a subject to be tested using a method according to any one of Claims 1 to 35 or 47.

50. A method for selecting a pair of cattle for breeding comprising determining the MHC type of a first breeding pair member using a method according to Claim 48, and selecting a second breeding pair member having a MHC type compatible with the MHC type of a first breeding pair member.

51. A method for selecting cattle for breeding comprising determining the MHC type of a population of candidate breeding pair members using a method according to Claim 48 and selecting candidate breeding pair members for breeding having a desired MHC type.

52. A method for selecting semen for use in artificial insemination in cattle comprising determining the MHC type of a candidate semen using a method according to Claim 48 and selecting semen having a desired MHC type.

53. A method for selecting donor of semen for use in artificial msemination in cattle comprising determining the MHC type of a candidate semen donor using a method according to Claim 48 and selecting a semen donor having a desired MHC type.

54. A method for selecting an embryo for use in artificial insemination in cattle comprising determining the MHC type of a candidate embryo using a method according to Claim 48 and selecting a candidate embryo having a desired MHC type.

55. A method according to of Claims 48 to 54 wherein the results ofthe methods are furnished in an intelligible format.

56. A method according to Claim 55 wherein the results are recorded or stored on an information carrier.

57. A kit of parts for use in a method according to any one of Clahns 1 to 35 or 47 to 56 comprising an isolated polynucleotide according to Claim 36 or 37 and an isolated polynucleotide according to Claim 38 or 39.

58. A kit of parts according to Claim 57 further comprising an isolated polynucleotide according to Claim 40 or 41.

59. A kit of parts according to Claim 57 or 58 comprising an isolated polynucleotide according to any one of Claims 42 or 43.

60. A kit of parts according to any one of Claims 57 to 59 comprising:

(a) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 1, an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 2 and an isolated polynucleotide comprising or consisting ofthe nucleotide sequence SEQ ID NO: 3 or 4; and/or (b) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 5, an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 6 and an isolated polynucleotide comprising or consisting ofthe nucleotide sequence SEQ ID NO: 7; and/or (c) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 8, an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 9 and an isolated polynucleotide comprising or consisting ofthe nucleotide sequence SEQ ID NO: 10 or 1; and/or (d) an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 12 or 14, an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 13 and an isolated polynucleotide comprising or consisting of the nucleotide sequence SEQ ID NO: 15 or 16.

61. A kit of parts according to any one of Claims 57to 60 further comprising one or more reagents for performing PCR.

62. A kit of parts according to any one of Claims 57 to 61 further comprising a control nucleic acid sample.

63. A kit of parts according to any one of Claims 57 to 62 further comprising instructions for performing a method according to any one of Claims 1 to 35 or 47 to 56.

64. A method for identifying major histocompatibility complex (MHC) genes and/or allelic variants thereof in cattle substantially as hereinbefore described with reference to the description.

65. A kit of parts substantially as hereinbefore described with reference to the description.