MHC ALLELES

Field of the invention

The present invention relates to methods of identifying MHC alleles present in a canine genome.

Background of the invention

Canine MHC molecules are known as dog leucocyte antigens (DLA). The DLA complex resides on chromosome 12, and DLA can be divided into class I, II and III regions.

Summary of the invention

Previously known methods for identifying canine MHC alleles include sequencing and reference strand-mediated conformation analysis (RSCA). However, both of these techniques are time consuming and expensive to perform on a routine basis. It would therefore be advantageous to provide an alternative method of identifying canine MHC alleles.

The present inventors have identified single nucleotide polymorphisms (SNPs) that can be used to uniquely identify each canine MHC allele. This has allowed the development of a SNP -based test for identification of canine MHC alleles. Furthermore, the present inventors have identified a collection of previously unknown canine MHC alleles. Accordingly, the invention provides a method for identifying one or more MHC alleles present in a dog, the method comprising:

(a) determining the nucleotide present at the or each polymorphic position specified for the one or more MHC alleles in any one of Tables 4 to 6 or determining the nucleotide(s) present at a polymorphic position(s) in linkage disequilibrium with one or more polymorphic positions specified in Tables 4 to 6; and

(b) identifying therefrom the presence or absence of one or more MHC alleles in the dog.

The invention further provides: a probe, primer or antibody which is capable of detecting the or each polymorphism as defined herein; a kit for carrying out the method of the invention, comprising means for detecting the or each polymorphism as defined herein; a method of determining whether a dog is susceptible to an MHC allele-

related disorder, the method comprising:

(a) identifying the presence or absence of one or more MHC alleles in the dog by a method according to the invention; and

(b) determining therefrom whether the dog is susceptible to an MHC allele-related disorder. a method of preparing customised food for a dog which is susceptible to an MHC allele-related disorder, the method comprising:

(a) determining whether the dog is susceptible to an MHC allele-related disorder by a method of the invention; and

(b) preparing food suitable for the dog. use of a compound which is therapeutic for an MHC allele-related disorder in the manufacture of a medicament for the prevention or treatment of an MHC allele-related disorder in a dog that has been identified as being susceptible to an MHC allele-related disorder by a method according to the invention; a method of treating a dog for an MHC allele-related disorder, the method comprising administering to the dog an effective amount of a therapeutic compound which prevents or treats the disorder, wherein the dog has been identified as being susceptible to an MHC allele-related disorder by a method according to the invention; a database comprising information relating to MHC allele polymorphisms as set out in any one of Tables 4 to 6 and optionally their association with MHC allele-related disorder(s); a method for identifying one or more MHC alleles in a dog, the method comprising:

(a) inputting data of the nucleotide present at the or each polymorphic position specified for one or more MHC alleles in any one of Tables 4 to 6 to a computer system;

(b) comparing the data to a computer database as defined herein; and

(c) identifying on the basis of the comparison the presence or absence of one or more MHC alleles in the dog; a computer program comprising program code means for performing all the steps of a method of the invention when said program is run on a computer; a computer program product comprising program code means stored on a computer readable medium for performing a method of the invention when said program product is run on a computer;

a computer program product comprising program code means on a carrier wave, which program code means, when executed on a computer system, instruct the computer system to perform a method according to the invention; a computer system arranged to perform a method according to the invention comprising:

(a) means for receiving data of the nucleotide present at the or each polymorphic position as specified in any one of Tables 4 to 6 in the dog;

(b) a module for comparing the data with a database as defined herein; and

(c) means for determining on the basis of said comparison the presence or absence of one or more MHC alleles. a method of preparing customised food for a dog which is susceptible to an MHC allele-related disorder, the method comprising:

(a) determining whether the dog is susceptible to an MHC allele-related disorder by a method according to the invention;

(b) electronically generating a customised dog food formulation suitable for the dog;

(c) generating electronic manufacturing instructions to control the operation of food manufacturing apparatus in accordance with the customised dog food formulation; and

(d) manufacturing the customised dog food according to the electronic manufacturing instructions. use of a computer system as defined herein to make a customised dog food product.

Description of the Figures

Figure 1 shows an apparatus of the invention.

Detailed description of the invention

It is known that certain characteristics and susceptibility to some diseases and disorders are correlated with the presence of particular MHC alleles or haplotypes. The method of the invention can therefore be further used to identify dogs that are susceptible to diseases that have a known association with one or more MHC alleles, referred to herein as an "MHC allele-related

disorder". Examples of disease susceptibilities that are linked to the presence of one or more MHC alleles or haplotypes are diabetes, hyperthyroidism and leishmaniasis.

A dog of any breed may be tested by a method of the present invention. The table below provides examples of dog breeds, wherein S = small, M = medium, L= large and XL = extra large. a) Hounds

b) Working Dogs

c)

e) Pastoral (Herding Group)

f)

g)

Detection of polymorphisms

The animal tested according to the present invention is a dog. The dog tested may be of any breed, or may be a mixed or crossbred dog, or an outbred dog (mongrel). The

polymorphisms detected are one or more of those set out in Tables 4 to 6. Typically, at least 5, such as at least 10, 20, 50, 100 or at least 200 of the polymorphisms set out in Tables 4 to 6 are detected. The detection of polymorphisms according to the invention may comprise contacting a polynucleotide or protein of the dog with a specific binding agent for a polymorphism and determining whether the agent binds to the polynucleotide or protein, wherein binding of the agent indicates the presence of the polymorphism, and lack of binding of the agent indicates the absence of the polymorphism.

The method is generally carried out in vitro on a sample from the dog. The sample typically comprises a body fluid and/or cells of the dog and may, for example, be obtained using a swab, such as a mouth swab. The sample may be a blood, urine, saliva, skin, cheek cell or hair root sample. The sample is typically processed before the method is carried out, for example DNA extraction may be carried out. The polynucleotide or protein in the sample may be cleaved either physically or chemically, for example using a suitable enzyme. In one embodiment the part of polynucleotide in the sample is copied or amplified, for example by cloning or using a PCR based method prior to detecting the polymorphism.

In the present invention, any one or more methods may comprise determining the presence or absence of one or more polymorphisms in the dog. The polymorphism is typically detected by directly determining the presence of the polymorphic sequence in a polynucleotide or protein of the dog. Such a polynucleotide is typically genomic DNA, mRNA or cDNA. The polymorphism may be detected by any suitable method such as those mentioned below.

A specific binding agent is an agent that binds with preferential or high affinity to the protein or polypeptide having the polymorphism but does not bind or binds with only low affinity to other polypeptides or proteins. The specific binding agent may be a probe or primer. The probe may be a protein (such as an antibody) or an oligonucleotide. The probe may be labelled or may be capable of being labelled indirectly. The binding of the probe to the polynucleotide or protein may be used to immobilise either the probe or the polynucleotide or protein.

Generally in the method, determination of the binding of the agent to the polymorphism can be carried out by determining the binding of the agent to the polynucleotide or protein of the dog. However in one embodiment the agent is also able to bind the corresponding wild-type sequence, for example by binding the nucleotides or amino acids which flank the polymorphic position, although the manner of binding to the wild-type sequence will be detectably different to the binding of a polynucleotide or protein containing the polymorphism.

The method may be based on an oligonucleotide ligation assay in which two oligonucleotide probes are used. These probes bind to adjacent areas on the polynucleotide which contains the polymorphism, allowing after binding the two probes to be ligated together by an appropriate ligase enzyme. However the presence of single mismatch within one of the probes may disrupt binding and ligation. Thus ligated probes will only occur with a polynucleotide that contains the polymorphism, and therefore the detection of the ligated product may be used to determine the presence of the polymorphism.

In one embodiment the probe is used in a heteroduplex analysis based system, hi such a system when the probe is bound to polynucleotide sequence containing the polymorphism it forms a heteroduplex at the site where the polymorphism occurs and hence does not form a double strand structure. Such a heteroduplex structure can be detected by the use of single or double strand specific enzyme. Typically the probe is an RNA probe, the heteroduplex region is cleaved using RNAase H and the polymorphism is detected by detecting the cleavage products.

The method may be based on fluorescent chemical cleavage mismatch analysis which is described for example in PCR Methods and Applications 3, 268-71 (1994) and Proc. Natl. Acad. ScL 85, 4397-4401 (1998).

In one embodiment a PCR primer is used that primes a PCR reaction only if it binds a polynucleotide containing the polymorphism, for example a sequence- or allele-specifϊc PCR system, and the presence of the polymorphism may be determined by the detecting the PCR product. Preferably the region of the primer which is complementary to the polymorphism is at or near the 3' end of the primer. The presence of the polymorphism may be determined using a fluorescent dye and quenching agent-based PCR assay such as the Taqman PCR detection system.

The specific binding agent may be capable of specifically binding the amino acid sequence encoded by a polymorphic sequence. For example, the agent may be an antibody or antibody fragment. The detection method may be based on an ELISA system. The method may be an RFLP based system. This can be used if the presence of the polymorphism in the polynucleotide creates or destroys a restriction site that is recognised by a restriction enzyme.

The presence of the polymorphism may be determined based on the change which the presence of the polymorphism makes to the mobility of the polynucleotide or protein during gel electrophoresis. In the case of a polynucleotide single-stranded conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DDGE) analysis may be used. In another method of detecting the polymorphism a polynucleotide comprising the polymorphic region is

sequenced across the region which contains the polymorphism to determine the presence of the polymorphism.

The presence of the polymorphism may be detected by means of fluorescence resonance energy transfer (FRET). In particular, the polymorphism may be detected by means of a dual hybridisation probe system. This method involves the use of two oligonucleotide probes that are located close to each other and that are complementary to an internal segment of a target polynucleotide of interest, where each of the two probes is labelled with a fiuorophore. Any suitable fluorescent label or dye may be used as the fiuorophore, such that the emission wavelength of the fiuorophore on one probe (the donor) overlaps the excitation wavelength of the fiuorophore on the second probe (the acceptor). A typical donor fiuorophore is fluorescein (FAM), and typical acceptor fluorophores include Texas red, rhodamine, LC-640, LC-705 and cyanine 5 (Cy5).

In order for fluorescence resonance energy transfer to take place, the two fluorophores need to come into close proximity on hybridisation of both probes to the target. When the donor fiuorophore is excited with an appropriate wavelength of light, the emission spectrum energy is transferred to the fiuorophore on the acceptor probe resulting in its fluorescence. Therefore, detection of this wavelength of light, during excitation at the wavelength appropriate for the donor fiuorophore, indicates hybridisation and close association of the fluorophores on the two probes. Each probe may be labelled with a fiuorophore at one end such that the probe located upstream (5') is labelled at its 3' end, and the probe located downstream (3') is labelled at is 5' end. The gap between the two probes when bound to the target sequence may be from 1 to 20 nucleotides, preferably from 1 to 17 nucleotides, more preferably from 1 to 10 nucleotides, such as a gap of 1, 2, 4, 6, 8 or 10 nucleotides.

The first of the two probes may be designed to bind to a conserved sequence of the gene adjacent to a polymorphism and the second probe may be designed to bind to a region including one or more polymorphisms. Polymorphisms within the sequence of the gene targeted by the second probe can be detected by measuring the change in melting temperature caused by the resulting base mismatches. The extent of the change in the melting temperature will be dependent on the number and base types involved in the nucleotide polymorphisms.

Polymorphisms which are in linkage disequilibrium with each other in a population are typically found together on the same chromosome. Typically one is found at least 30% of the times, for example at least 40 %, at least 50%, at least 70% or at least 90%, of the time the other is found on a particular chromosome in individuals in the population. Thus a

polymorphism which is not a functional susceptibility polymorphism, but is in linkage disequilibrium with a functional polymorphism, may act as a marker indicating the presence of the functional polymorphism.

Polymorphisms which are in linkage disequilibrium with the polymorphisms mentioned herein are typically located within 500kb, preferably within 400kb, within 200kb, within 100kb, within 50kb, within 10kb, within 5kb, within 1 kb, within 500bp, within lOObp, within 50bp or within 1 Obp of the polymorphism.

Polynucleotides

The polynucleotide is typically at least 10, 15, 20, 30, 50, 100, 200 or 500 bases long, such as at least or up to lkb, 10kb, 100kb, 1000 kb or more in length. The polynucleotide will typically comprise flanking nucleotides on one or both sides of (5 ¹ or 3' to) the polymorphism, for example at least 2, 5, 10, 15 or more flanking nucleotides in total or on each side. Typically, the polynucleotide will be at least 95%, preferably at least 99%, even more preferably at least 99.9% identical to the reference polynucleotide sequences. Such numbers of substitutions and/or insertions and/or deletions and/or percentage identity may be taken over the entire length of the polynucleotide or over 50, 30, 15, 10 or less flanking nucleotides in total or on each side.

The polynucleotide may be RNA or DNA, including genomic DNA, synthetic DNA or cDNA. The polynucleotide may be single or double stranded. The polynucleotide may comprise synthetic or modified nucleotides, such as methylphosphonate and phosphorothioate backbones or the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.

A polynucleotide of the invention may be used as a primer, for example for PCR, or a probe. A polynucleotide or polypeptide of the invention may carry a revealing label. Suitable labels include radioisotopes such as ³² P or ³⁵ S, fluorescent labels, enzyme labels or other protein labels such as biotin.

The invention also provides expression vectors that comprise polynucleotides of the invention and are capable of expressing a polypeptide of the invention. Such vectors may also comprise appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for protein expression. Thus the coding sequence in the vector is operably linked to such elements so that they provide for expression of the coding sequence

(typically in a cell). The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.

The vector may be for example plasmid, virus or phage vector. Typically the vector has an origin of replication. The vector may comprise one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example in a method of gene therapy.

Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. Mammalian promoters, such as β-actin promoters, may be used. Tissue- specific promoters are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR).

The vector may further include sequences flanking the polynucleotide giving rise to polynucleotides which comprise sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of the polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for delivering the polynucleotides of the invention to a mammalian cell. Other examples of suitable viral vectors include herpes simplex viral vectors and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. Gene transfer techniques using these viruses are known to those skilled in the art. Retrovirus vectors for example may be used to stably integrate the polynucleotide giving rise to the polynucleotide into the host genome. Replication-defective adenovirus vectors by contrast remain episomal and therefore allow transient expression.

Polynucleotides of the invention may be used as a probe or primer which is capable of

selectively binding to a polymorphism. Preferably the probe or primer is capable of selectively binding to a reference polynucleotide sequence. The invention thus provides a probe or primer for use in a method according to the invention, which probe or primer is capable of selectively detecting the presence of a polymorphism. Preferably the probe is isolated or recombinant nucleic acid. It may correspond to or be antisense to the reference polynucleotide sequence. The probe may be immobilised on an array, such as a polynucleotide array.

Such primers, probes and other fragments will preferably be at least 10, preferably at least 15 or at least 20, for example at least 25, at least 30 or at least 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100 or 150 nucleotides in length. Probes and fragments can be longer than 150 nucleotides in length, for example up to 200, 300, 400, 500, 600, 700 nucleotides in length, or even up to a few nucleotides, such as five or ten nucleotides, short of a full length polynucleotide sequence of the invention.

Homologues

Homologues of polynucleotide or protein sequences are referred to herein. Such homologues typically have at least 70% homology, preferably at least 80, 90%, 95%, 97% or 99% homology, for example over a region of at least 15, 20, 30, 100 more contiguous nucleotides or amino acids. The homology may be calculated on the basis of nucleotide or amino acid identity (sometimes referred to as "hard homology").

For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology (for example used on its default settings) (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent or corresponding sequences (typically on their default settings), for example as described in Altschul S. F. (1993) J MoI Evol 36:290-300; Altschul, S, F et al (1990) J MoI Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score

falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sd. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sd. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two polynucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The homologous sequence typically differs by at least 1, 2, 5, 10, 20 or more mutations, which may be substitutions, deletions or insertions of nucleotide or amino acids. These mutations may be measured across any of the regions mentioned above in relation to calculating homology. In the case of proteins the substitutions are preferably conservative substitutions. These are defined according to the following Table. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

Shorter polypeptide sequences are also within the scope of the invention. For example, a fragment of a polypeptide sequence of the invention is typically at least 10, 15, 20, 30, 40, 50,

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60, 70, 80, 100, 150 or 200 amino acids in length. Polypeptides of the invention may be chemically modified, for example post-translationally modified. The polypeptides may be glycosylated or comprise modified amino acid residues. Such modified polypeptides fall within the scope of the term "polypeptide" of the invention.

The polypeptides, polynucleotides, vectors, cells or antibodies of the invention maybe present in an isolated or substantially purified form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98% or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.

It is understood that any of the above features that relate to polynucleotides and proteins may also be a feature of the other polypeptides and proteins mentioned herein, such as the polypeptides and proteins used in the screening and therapeutic aspects of the invention, hi particular such features may be any of the lengths, modifications and vectors forms mentioned above.

Detector antibodies

The invention also provides detector antibodies that are specific for a polypeptide of the invention. A detector antibody is specific for one polymorphism, but does not bind to any other polymorphism. The detector antibodies of the invention are for example useful in purification, isolation or screening methods involving immunoprecipitation techniques.

Antibodies may be raised against specific epitopes of the polypeptides of the invention. An antibody, or other compound, "specifically binds" to a polypeptide when it binds with preferential or high affinity to the protein for which it is specific but does substantially bind not bind or binds with only low affinity to other polypeptides. A variety of protocols for competitive binding or immunoradiometric assays to determine the specific binding capability of an antibody are well known in the art (see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Such immunoassays typically involve the formation of complexes between the specific protein and its antibody and the measurement of complex formation.

For the purposes of this invention, the term "antibody", unless specified to the contrary, includes fragments which bind a polypeptide of the invention. Such fragments include Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies. Furthermore, the antibodies and fragment thereof may be chimeric antibodies, CDR-grafted antibodies or humanised antibodies.

1 /

Antibodies may be used in a method for detecting polypeptides of the invention in a biological sample (such as any such sample mentioned herein), which method comprises:

I providing an antibody of the invention;

II incubating a biological sample with said antibody under conditions which allow for the formation of an antibody-antigen complex; and

III determining whether antibody-antigen complex comprising said antibody is formed. Antibodies of the invention can be produced by any suitable method. Means for preparing and characterising antibodies are well known in the art, see for example Harlow and Lane (1988) "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. For example, an antibody may be produced by raising antibody in a host animal against the whole polypeptide or a fragment thereof, for example an antigenic epitope thereof, herein after the "immunogen". The fragment may be any of the fragments mentioned herein (typically at least 10 or at least 15 amino acids long).

A method for producing a polyclonal antibody comprises immunising a suitable host animal, for example an experimental animal, with the immunogen and isolating immunoglobulins from the animal's serum. The animal may therefore be inoculated with the immunogen, blood subsequently removed from the animal and the IgG fraction purified. A method for producing a monoclonal antibody comprises immortalising cells which produce the desired antibody. Hybridoma cells may be produced by fusing spleen cells from an inoculated experimental animal with tumour cells (Kohler and Milstein (1975) Nature 256, 495-497).

An immortalized cell producing the desired antibody may be selected by a conventional procedure. The hybridomas may be grown in culture or injected intraperitoneally for formation of ascites fluid or into the blood stream of an allogenic host or immunocompromised host. Human antibody may be prepared by in vitro immunisation of human lymphocytes, followed by transformation of the lymphocytes with Epstein-Barr virus.

For the production of both monoclonal and polyclonal antibodies, the experimental animal is suitably a goat, rabbit, rat, mouse, guinea pig, chicken, sheep or horse. If desired, the immunogen may be administered as a conjugate in which the immunogen is coupled, for example via a side chain of one of the amino acid residues, to a suitable carrier. The carrier molecule is typically a physiologically acceptable carrier. The antibody obtained may be isolated and, if desired, purified.

Detection kit

The invention also provides a kit that comprises means for determining the presence or absence of one or more canine MHC polymorphism(s). In particular, such means may include a specific binding agent, probe, primer, pair or combination of primers, or antibody, including an antibody fragment, as defined herein which is capable of detecting or aiding detection of a polymorphism. The primer or pair or combination of primers may be sequence specific primers which only cause PCR amplification of a polynucleotide sequence comprising the polymorphism to be detected, as discussed herein. The kit may also comprise a specific binding agent, probe, primer, pair or combination of primers, or antibody which is capable of detecting the absence of the polymorphism. The kit may further comprise buffers or aqueous solutions.

The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments of the method mentioned above to be carried out. Such reagents or instruments may include one or more of the following: a means to detect the binding of the agent to the polymorphism, a detectable label such as a fluorescent label, an enzyme able to act on a polynucleotide, typically a polymerase, restriction enzyme, ligase, RNAse H or an enzyme which can attach a label to a polynucleotide, suitable buffer(s) or aqueous solutions for enzyme reagents, PCR primers which bind to regions flanking the polymorphism as discussed herein, a positive and/or negative control, a gel electrophoresis apparatus, a means to isolate DNA from sample, a means to obtain a sample from the individual, such as swab or an instrument comprising a needle, or a support comprising wells on which detection reactions can be carried out. The kit may be, or include, an array such as a polynucleotide array comprising the specific binding agent, preferably a probe, of the invention. The kit typically includes a set of instructions for using the kit.

Treatment of MHC-related disorders

The invention also provides a method of treating a dog for an MHC-related disorder, the method comprising identifying a dog which is susceptible to an MHC-related disorder by a method of the invention, and administering to the dog an effective amount of a therapeutic agent which treats the MHC-related disorder. The MHC allele-related disorder may be any disease or disorder mentioned herein, for example, diabetes, hyperthyroidism and leishmaniasis.

The therapeutic agent may be administered in various manners such as orally, intracranially, intravenously, intramuscularly, intraperitoneally, intranasally, intrademally, and subcutaneously. The pharmaceutical compositions that contain the therapeutic agent will normally be formulated with an appropriate pharmaceutically acceptable carrier or diluent

depending upon the particular mode of administration being used. For instance, parenteral formulations are usually injectable fluids that use pharmaceutically and physiologically acceptable fluids such as physiological saline, balanced salt solutions, or the like as a vehicle. Oral formulations, on the other hand, may be solids, for example tablets or capsules, or liquid solutions or suspensions. In a preferred embodiment, the therapeutic agent is administered to the dog in its diet, for example in its drinking water or food.

The amount of therapeutic agent that is given to a dog will depend upon a variety of factors including the condition being treated, the nature of the dog under treatment and the severity of the condition under treatment. A typical daily dose is from about 0.1 to 50 mg per kg, preferably from about 0.1mg/kg to lOmg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the dog to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g.

Customised food

In one aspect, the invention relates to a customised food for a dog, which is customised based on the canine MHC alleles present. Such a food may be in the form of, for example, wet pet foods, semi-moist pet foods, dry pet foods and pet treats. Wet pet food generally has a moisture content above 65%. Semi-moist pet food typically has a moisture content between 20- 65% and can include humectants and other ingredients to prevent microbial growth. Dry pet food, also called kibble, generally has a moisture content below 20% and its processing typically includes extruding, drying and/or baking in heat. The ingredients of a dry pet food generally include cereal, grains, meats, poultry, fats, vitamins and minerals. The ingredients are typically mixed and put through an extruder/cooker. The product is then typically shaped and dried, and after drying, flavours and fats may be coated or sprayed onto the dry product.

Accordingly, the present invention enables the preparation of customised food suitable for a dog with a particular MHC allele or combination of alleles (haplotype). In particular, the food may be customised for a dog which is susceptible to an MHC allele-related disorder, wherein the customised dog food formulation comprises ingredients that prevent or alleviate the MHC allele-related disorder, and/or does not comprise components that contribute to or aggravate the MHC allele-related disorder. Such ingredients may be any of those known in the art to prevent or alleviate an MHC allele-related disorder. The preparation of customised dog food may be carried out by electronic means, for example by using a computer system.

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The present invention also relates to a method of providing a customised dog food, comprising providing food suitable for a dog with a particular MHC allele or alleles to the dog, the dog's owner or the person responsible for feeding the dog, wherein the MHC allele has been identified by a method of the invention. In one aspect of the invention, the customised food is made to inventory and supplied from inventory, i.e. the customised food is pre-manufactured rather than being made to order. Therefore according this apect of the invention the customised food is not specifically designed for one particular dog but instead is suitable for more than one dog. Alternatively, the customised food may be suitable for a sub-group of dogs with a particular MHC allele, such as dogs of a particular breed, size or lifestage. In another embodiment, the food may be customised to meet the nutritional requirements of an individual dog.

Bioinformatics

The sequences of the MHC alleles may be stored in an electronic format, for example in a computer database. Accordingly, the invention provides a database comprising information relating to MHC polymorphic sequences. The database may include further information about the polymorphism, for example the frequency of the polymorphism in the population or in each breed. In one aspect of the invention, the database further comprises information regarding the food components which are suitable and the food components which are not suitable for dogs who possess a particular MHC allele.

A database as described herein may be used to identify the MHC allele present in a dog. Such a determination may be carried out by electronic means, for example by using a computer system (such as a PC). Typically, the determination will be carried out by inputting genetic data from the dog to a computer system; comparing the genetic data to a database comprising information relating to MHC polymorphisms; and on the basis of this comparison, identifying the or each MHC allele present in the dog.

The invention also provides a computer program comprising program code means for performing all the steps of a method of the invention when said program is run on a computer. Also provided is a computer program product comprising program code means stored on a computer readable medium for performing a method of the invention when said program is run on a computer. A computer program product comprising program code means on a carrier wave that, when executed on a computer system, instruct the computer system to perform a method of the invention is additionally provided.

As illustrated in Figure 1, the invention also provides an apparatus arranged to perform a method according to the invention. The apparatus typically comprises a computer system, such as a PC. In one embodiment, the computer system comprises: means 20 for receiving genetic data from the dog; a module 30 for comparing the data with a database 10 comprising information relating to MHC polymorphisms; and means 40 for identifying on the basis of said comparison the MHC allele present in the dog.

Food manufacturing

In one embodiment of the invention, the manufacture of a customised dog food maybe controlled electronically. Typically, information relating to the or each MHC allele present in a dog may be processed electronically to generate a customised dog food formulation. The customised dog food formulation may then be used to generate electronic manufacturing instructions to control the operation of food manufacturing apparatus. The apparatus used to carry out these steps will typically comprise a computer system, such as a PC, which comprises means 50 for processing the nutritional information to generate a customised dog food formulation; means 60 for generating electronic manufacturing instructions to control the operation of food manufacturing apparatus; and a food product manufacturing apparatus 70.

The food product manufacturing apparatus used in the present invention typically comprises one or more of the following components: container for dry pet food ingredients; container for liquids; mixer; former and/or extruder; cut-off device; cooking means (e.g. oven); cooler; packaging means; and labelling means. A dry ingredient container typically has an opening at the bottom. This opening may be covered by a volume-regulating element, such as a rotary lock. The volume-regulating element may be opened and closed according to the electronic manufacturing instructions to regulate the addition of dry ingredients to the pet food.

Dry ingredients typically used in the manufacture of pet food include corn, wheat, meat and/or poultry meal. Liquid ingredients typically used in the manufacture of pet food include fat, tallow and water. A liquid container may contain a pump that can be controlled, for example by the electronic manufacturing instructions, to add a measured amount of liquid to the pet food.

In one embodiment, the dry ingredient container(s) and the liquid container(s) are coupled to a mixer and deliver the specified amounts of dry ingredients and liquids to the mixer. The mixer may be controlled by the electronic manufacturing instructions. For example, the duration or speed of mixing may be controlled. The mixed ingredients are typically then delivered to a former or extruder. The former/extruder may be any former or extruder known in the art that can be used to shape the mixed ingredients into the required shape. Typically, the

mixed ingredients are forced through a restricted opening under pressure to form a continuous strand. As the strand is extruded, it may be cut into pieces (kibbles) by a cut-off device, such as a knife. The kibbles are typically cooked, for example in an oven. The cooking time and temperature may be controlled by the electronic manufacturing instructions. The cooking time may be altered in order to produce the desired moisture content for the food. The cooked kibbles may then be transferred to a cooler, for example a chamber containing one or more fans.

The food manufacturing apparatus may comprise a packaging apparatus. The packaging apparatus typically packages the food into a container such as a plastic or paper bag or box. The apparatus may also comprise means for labelling the food, typically after the food has been packaged. The label may provide information such as: ingredient list; nutritional information; date of manufacture; best before date; weight; and species and/or breed(s) for which the food is suitable.

The invention is illustrated by the following Examples:

Example 1

Materials and Methods

MHC genotyping for DLA-DRBl, DQAl and DQBl

Dogs were characterised for three DLA class II loci using either sequence based typing (SBT) (Kennedy et al. 2002; Kennedy et al. 1998) or Reference Strand-mediated Conformation Analysis (RSCA) (Kennedy et al. 2005).

All PCR reactions are performed with 25ng DNA in a 25 μl reaction containing Ix PCR buffer as supplied by Qiagen (with no extra magnesium), Q solution (Qiagen), final concentrations of 0.1 μM for each primer, 200μM each dNTP, with 2 units of Taq polymerase, (Qiagen HotStarTaq). A negative control containing no DNA template should be included in each run of amplifications to identify any contamination.

Primers used were: DRBF forward: gat ccc ccc gtc ccc aca g, DRBR3 reverse: cgc ccg ctg cgc tea, DQAinl forward: taa ggt tct ttt etc cct ct, DQAIn2 reverse: gga cag att cag tga aga ga, DQBlB forward: etc act ggc ccg get gtc tc and DQBR2 reverse: cac etc gcc get gca acg tg. All primers are intronic and locus specific, and the product sizes are 303bp for DLA-DRBl, 345bp for DQAl and 300bp for DQBl .

A standard Touchdown PCR protocol was used for all amplifications, which consisted of an initial 15 minutes at 95 ⁰ C, 14 touch down cycles of 95 ⁰ C for 30 seconds, followed by 1 minute annealing, starting at 62 ⁰ C (DRBl), 54 ⁰ C (DQAl) 73°C (DQBl) and reducing by 0.5 ⁰ C each cycle, and 72°C for 1 minute. Then 20 cycles of 95°C for 30 seconds, 55°C (DRBl), 47°C (DQAl) 66°C (DQBl) for 1 minute, 72 ⁰ C for 1 minute plus a final extension at 72°C for 10 minutes.

To check for the presence of a product, 5μl was run on a 2% agarose gel. No purification was required for RSCA. However, this was required SBT: 2 units of shrimp alkaline phosphatase (USB) and 10 units of Exol (New England Biolabs) were added to 5μl of PCR product. The mixture was incubated for 1 hour at 37°C, then for 15 minutes at 80 ⁰ C.

To perform RSCA, FLRs were generated, using a range of DLA-DRBl alleles from the domestic dog and grey wolf. The FLRs were produced by PCR using cloned alleles as templates and a 5'-FAM22 labelled forward primer. In order to increase the proportion of the labelled reference strand in the reaction, the primer proportions were altered to 0.5μM FAM22-labelled forward primer and 0.1 μM reverse unlabelled primer. All other aspects of the PCR reaction remained the same. This single stranded-biased FLR was used to increase the heights of the FLR-allele heteroduplex peaks relative to the homoduplex peaks in subsequent RSCA (data not presented). AU the resulting FLRs were diluted 1:30 in water before use in the hybridisation reactions.

In order to form duplexes between test samples and FLRs, 2μl of diluted FLR and 2μl of test sample PCR product were mixed in a 96 well plate and incubated in a thermal cycler at 95 ⁰ C for 10 minutes, ramped down to 55°C at l°C/second, 55°C for 15 minutes and 4 ⁰ C for 15 minutes. The plate was stored at 4°C until required. Subsequently, 8μl distilled water were added to each hybridisation reaction, and then 2μl were mixed with 4.8μl water and 0.2μl Genescan Rox-500 size standards (Applied Biosystems), in a 384 well plate. These samples were run on an ABI 3100 DNA analyser, using 50cm capillary arrays, 4% Genescan non-denaturing polymer (Applied Biosystems) and data collected using matrix Dye set D. The conditions were: injection voltage 15kV, injection time 15 seconds, run voltage 15kV, run temperature 3O ⁰ C. Each run took 35 minutes. The data were analysed using software programs "Genescan" and "Genotyper" (Applied Biosystems). Genescan was used to assign sizes to each peak, based on the ROX-500 standards. Using Genotyper, allele peaks formed by the control samples were assigned to "bins" for each FLR used. The bins were exported to an in-house program, (Martin A, unpublished), which assigned the alleles for each sample.

The allelic names and sequences for each allele are shown below:

DLA dqal . L12 , exon 2 (nucleotides 15-260)

>DQAl*00101

GAC CAT GTT GCC AAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT AGA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ATA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC

CAA ACT GCT GCT ACC AAT

>DQAl*00201

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ACA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC

AAA ACT GCT GCT ACC AAT

>DQAl*00301

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCC AGA GCA AAA CAA AAC TTG AAC ATC CTG ACT AAA AGT TCC AAC

CAA ACT GCT GCT ACC AAT

>DQAl*00401

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ATA AAA CAA AAC TTG AAC ATC CTG ACT AAA AGG TCC AAC

CAA ACT GCT GCT ACC AAT

>DQAl*005011

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TTC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ACA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC

AAA ACT GCT GCT ACC AAT

>DQAl*005012

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TTC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCG

CTG AGA AAC TTG GCT ATA ACA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC

AAA ACT GCT GCT ACC AAT

>DQAl*00601

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT AGA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ATA AAA CAA AAC TTG AAC ATC CTG ACT AAA AGG TCC AAC

CAA ACT GCT GCT ACC AAT

>DQAl*00701

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ACA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC

CAA ACT GCT GCT ACC AAT

>DQAl*00801

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCC AGA GCA AAA CAA AAC TTG AAC ATC CTG ACT AAA AGG TCC AAC

CAA ACT GCT GCT ACC AAT

>DQAl*00901

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG TTC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT AGA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ATA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC CAA ACT GCT GCT ACC AAT >DQAl*01001

ACTGCTGCTACCAAT >DQAl*01101

ACTGCTGCTACCAAT >DQAl*012011

TTGACCCACAGGGTGCACTGAGAAACTTGGCTATAGCAAAACAAAACTTGAACATCA TGACTAAAAGGTCCAACCAA

ACTGCTGCTACCAAT

>dqal*012012

ACTGCTGCTACCAAT >DQAl*01301 GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT AGA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ACA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC AAA ACT GCT GCT ACC AAT >DQAl*014011 GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT AGA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ATA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC CAA ACT GCT GCT ACC AAT >DQA1*O14O12 GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG TAC ACA CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT AGA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ATA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC CAA ACT GCT GCT ACC AAT >DQAl*01501

ACTGCTGCTACCAAT

>07vl

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC TCT GGC CAG

TTC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG GAC CTG GAG AAG AAG GAA

ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA TTT ACA AGT TTT GAC CCA CAG GGT GCA

CTG AGA AAC TTG GCT ATA ACA AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC

CAA ACT GCT GCT ACC AAT

ACTGCTGCTACCAAT >dqa383-ll

Zb

TTGACCCACAGGGTGCACTGAGAAACTTGGCCATAACAAAACAAAACTTGAACATCA TGACTAAAAGGTCCAACAAA

ACTGCTGCTACCAAT

>DQAl*01601

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC

TCT GGC CAG TAC ACA CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG

GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA

TTT ACA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ATA

AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC CAA ACT GCT GCT

ACC AAT

>DQAl*01602

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC

TCT GGC CAG TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG

GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA

TTT ACA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ATA

AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC CAA ACT GCT GCT

ACC AAT

>DQA/M/LO51

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC

TCT GGC CAG TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG

GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA

TTT ACA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA GCA

AAA CAA AAC TTG AAC ATC CTG ACT AAA AGT TCC AAC CAA ACT GCT GCT

ACC AAT

>DQA/W53/B

GAC CAT GTT GCC aAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC

TCT GGC CAG TaC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG

GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA

TTT ACA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA AtA

AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC cAA ACT GCT GCT

ACC AAT

>DQAl*01701

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC

TCT GGC CAG TAC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG

GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA

TTT GCA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT AGA GCA

AAA CAA AAC TTG AAC ATC CTG ACT AAA AGT TCC AAC CAA ACT GCT GCT

ACC AAT

>DQA/COY954A

GAC CAT GTT GCC TAC TAC GGC ATA AAT GTC TAC CAG TCT TAC GGT CCC

TCT GGC CAG TTC ACC CAT GAA TTT GAT GGC GAT GAG GAG TTC TAC GTG

GAC CTG GAG AAG AAG GAA ACT GTC TGG CGG CTG CCT GTG TTT AGC ACA

TTT ACA AGT TTT GAC CCA CAG GGT GCA CTG AGA AAC TTG GCT ATA ATA

AAA CAA AAC TTG AAC ATC ATG ACT AAA AGG TCC AAC CAA ACT GCT GCT

ACC AAT

>hcdqa-lDM

GACCATGTTGC

CGATGAGGAG1

TTGACCCACAGGGTGCACTGAGAAACTTGGCTATAgCAAAACAAAACTTGAACATCA TGACTAAAAGGTCCAACAAA

ACTGCTGCTACCAAT

>awddqaθl

AcTGCtGCTaCCAaT

>dqa-lk-ew73

GACCATGTTGCCTACTACGGCATAAATGTCTACCAGTCTTACGGTCCCTCTGGCCAG TACACCCATGAATTTGATGG

TTGACCCACAGGGTGCACTGAGAAACTTGGCTATAgcAAAACAAAACTTGAACATCC TGACTAAAAGGTCCAACCAA ACTGCTGCTACCAAT

Il

DLA-DQBl (base 1 = base 16 of exon 2)

>DQBl*00101

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*00201

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*00301

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*00401

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*00501

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*00502

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*00701

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*008011

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*008012

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*00802

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*01101

GATTTCGTGTAC

CTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCT

CGGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGACCGGGTACGGGCCGAGCTGG ACACGGTGTGCAGACACAAC

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*01201

GATTTCGTGTTCCAGTTTAAGTTCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGCTTCTGGCGAGAGACAT

ACGCTGAGTACTGGAACGGGCAGAAGGAGCTCTTGGAGCAGAGGCGGGCCGAGCTGG ACACGGTGTGCAGACACAAC TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA

>DQBl* 01301

CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCG

ACGCTGAGTACTGGAACCCGCAGAAGGACGAGATGG^

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*01302

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*01303

ACGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGACCGGGTACGGGCCGAGCTGG ACACGGTGTGCAGACACAAC

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA

>DQBl*01304

GATTTCGTGTACCAGTTTAAGTTCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGCTTCTGACTAAATACAT

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQB1*O14O1

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*01501

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQB1*O16O1

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*01701

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*01801

TACGGGGTGGAAGAGCTCTCCACGTTGCAGCGGCGA >DQB1*O19O1

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQB1*O2OO1

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*02002

GATTTCGTGTAC

CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGT CACGGAGCTCGGGCGGCCCG

TCGCTGAGTACTGGAACGGGCAGAAGGAGATCTTGGAGCGGAAGCGGGCCGCGGTGG ACAGGGTGTGCAGACACAAC

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*02101

GATTTCGTGTACCAGTTTAAGGCCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGCTTCTGACGAGAAGCAT

CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGT CACGGAGCTCGGGCGGCCCG

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQB1*02201

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQB1*02301

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*02302

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*02401

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >jmadqb-ccahOO5

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*02601

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*02701

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*02801

TACGGGTTGGAAGAGCTCTACACGTTGCAGcGGcGA >DQBl*02901

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*03001

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*03101

GATTTCGTGTTC

CTATAACCGGG;

CGGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGACCGGGTACGGGCCGAGCTGG ACACGGTGTGCAGACACAAC

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*03201

CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCG ACGCTGAGTACTGGAACCGACAGAAGGACGAGATGG^

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*03301 GATTTCGTGTACCAGTTTAAGGCCGAGTGCTATTTCACCAACGGGACGGAGCGGGTGCGG CTTCTGACGAGAAGCAT

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*03401

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*03501

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*03601

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >dgbl*03701

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >lkdqbE18

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQB1*O39O1

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA >dqbrw2S9new

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >dqbw30new

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*03801

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*04001

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >dqb383-9

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA

>dqb-a32-008v

GATTTCGTGTACCi=

CTATAACCGGGAGG

ACGCTGAGTACTGGAACCcgCAGAAGGACGAGATGGACCGGGTACGGGCCGAGCTGG ACACGGTGTGCAGACACAAC

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>dqbwAnew

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*04101

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQBl*04201

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA

>dgb381-9

GATTTCGTGTTCCAGTTTAAGTTCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGCTTCTGgCTAgATACAT

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*04301

GATTTCGTGTaCCAGTTTAAGGgCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGCTTCTGgCtAaAtACAT

CTATAACCGGGAGGAGttCGTGCGCTTCGACAGCGAC

ACGCTGAGTACTGGAACGGGCAGAAGGAGaTCTTGG^

TACGGGgtGGAAGAGCTCtaCACGTTGCAGCGGCGA

>DQB/AA

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQB/BB

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA >DQB/DD

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQBl*04401

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA >DQB/H

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQB/I

GATTTCC

CTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCT

ACGCTGAGTACTGGAACGGGCAGAAGGAGTTCTTGGAGCGGGCGCGGGCCGAGGTGG ACACGGTGTGCAGACACAAC

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA

51 >DQB/J

CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCT

GGGCTGAGTACTGGAACGGGCAGAAGGAGATCTTGGS

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQBl*04501

acGCTGAGTACTGGAACGGGCAGAAGGAGtTCTTGGAGCGGgcGCGGGCCGcGgTGG ACACGGTGTGCAGACACAAC

TACGGGGTGGAAGAGCTCacCACGTTGCAGCGGCGA

>DQB/R gATTTcGTGTACCAGTTTAAGTTCGAGTGCTATTTCACCAACGGGACGGAGCGGGTGCGG CTTCTGACTAAATACAT

ACGCTGAGTACTGGAACccGCAGAAGGAcCagaTGGACCgGgtaCGGGCCGAGCTGG ACACGGTGTGCAGACACAAC

TACgGGgTGGAAGAGCTCTACACGTTGCAGCGGCGA

>DQB/S

GATTTCGTGTtCCAGTGTAAGGgCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGcTTCTGaCTAAATACAT ggGCTGAGTACTGGAACccGCAGAAGGAcCagaTGGAcCgGGtaCGGGCcgaGcTGGACA CGGTGTGCAGACACAAC

TACGGGtTGGAAGAGCTCTACACGTTGCAGCGGCGA

>DQB/U

CTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCg

ACGCTGAGTACTGGAACGGGCAGAAGGAGTTCTTGGS

TACGGGGTGGAAGAGCTCACCACGTTGCAGCGGCGA

>DQB/CVA307/B

GATTTCGTGTwCC;

CTATAACCGGGAGC

GGGCTGAGTACTGGAACCCGCAgAAGgACGAGATGGACcGGGTACgGGCCGAGCTGG ACACGGTGTGCAGACACAAC

TACGGGgTGGAAGAGCTCTACACGTTGCAGCGGCGA

>dqbIW001

TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA

>dqbl*03602

GATTTCGTGTTCCAGTATAAGGCCGAGTGCTATTTCACCAACGGGACGGAGC

GGGTGCGGCTTCTGACTAAATACATCTATAACCGGGAGGAGTTCGTGCGCTT

CGACAGCGACGTGGGGGAGTACCGGGCGGTCACGGAGCTCGGGCGGCCCGA

CGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGACCGGGTACGGGCCGA

GCTGGACACGGTGTGCAGACACAACTACGGGGTGGAAGAGCTCTACACGTTG

CAGCGGCGA

>dqbl*03603

GATTTCGTGTTCCAGTATAAGGCCGAGTGCTATTTCACCAACGGGACGGAGC

GGGTGCGGCTTCTGACTAAATACATCTATAACCGGGAGGAGTTCGTGCGCTT

CGACAGCGACGTGGGGGAGTACCGGGCGGTCACGGAGCTCGGGCGGCCCGA

CGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGACCGGGTACGGGCCGA

GCTGGACACGGTGTGCAGACACAACTACGGGAGGGAAGAGCTCACCACGTT

GCAGCGGCGA

>dqbl*00202

GATTTCGTGTTCCAGTATAAGGCCGAGTGCTATTTCACCAACGGGACGGAGC

GGGTGCGGCTTCTGACTAAATACATCTATAACCGGGAGGAGTTCGTGCGCTT

CGACAGCGACGTGGGGGAGTTCCGGGCGGTCACGGAGCTCGGGCGGCCCGA

CGCTGAGTACTGGAACCGACAGAAGGACGAGATGGACCGGGTACGGGCCGA

GCTGGACACGGTGTGCAGACACAACTACGGGGTGGAAGAGCTCTACACGTTG

CAGCGGCGA

>dqbl*04601

GATTTCGTGTACCAGTTTAAGTTCGAGTGCTATTTCACCAACGGGACGGAGCG

GGTGCGGCTTCTGACTAAATACATCTATAACCGGGAGGAGTTCGTGCGCTTC

GACAGCGACGTGGGGGAGTTCCGGGCGGTCACGGAGCTCGGGCGGCCCGAC

GCTGAGTACTGGAACCGACAGAAGGACGAGATGGACCGGGTACGGGCCGAG

CTGGACACGGTGTGCAGACACAACTACGGGAGGGAAGAGCTCACCACGTTG

CAGCGGCGA

>DQBl*04701

GATTTCGTGTTCCAGTGTAAGTTCGAGTGCTATTTCACCAACGGGACGGA GCGGGTGCGGTTTCTGGCTAAATACATCTATAACCGGGAGGAGTTCGTGC GCTTCGACAGCGACGTGGGGGAGTACCGGGCGGTCACGGAGCTCGGGCGG CCCGACGCTGAGTCCTGGAACGGGCAGAAGGAGCTCTTGGAGCAGGAGCG GGCAACGGTGGACACGGTGTGCAGACACAACTACGGGGTGGAAGAGCTCT ACACGTTGCAGCGGCGA >lk-awdl4 gATTtCGTgTaCcAGTTTAAGGGCGAGTGCTATTTCACCAACGGGACGGAGCGGGTGCGG CTTCTGACTAAACACAT CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGAC ACGCTGAGTACTGGAACCGGCAGAAGGACGAGGTGG-= TACGGGATGGAGGAGCTCACCACGTTGCAGCGGCGA >lk-awdl6 gATTtCgTGTaCcAGTTTAaGGGCGAGTGCTATTTCACCAACGGGACGGAGCGGGTGCGG TTCGTGGACAGATACAT CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGAC ACGCTGAGTACTGGAACCGGCAGAAGGACGAGGTGG^ TACGGGATGGAGGAGCTCACCACGTTGCAgCGGCGA >dqbO13+O17

GATTTCGTGTwCCAGTkTAAGkyCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGyTTCTGrCTAAATACAT CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGAC ACGCTGAGTmCTGGAACssGCAGAAGGAskwswTGGJ? TACGGGGTGGAAGAGCTCTACACGTTGCAGCGGCGA >dqb019+022

GATTTCGTGTwCCAGTkTAAGGsCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGyTTCTGrCTAAATACAT CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGAC CGGCTGAGTACTGGAACssGCAGAAGGASSWSWTGG^ TACGGGwkGGAAGAGCTCACCACGTTGCAGCGGCGA >dqb8061new

TACGGGAGGGAAGAGCTCACCACGTTGCAGCGGCGA

>dqb8062new

GATTTCGTGTACCAGTGTAAGGGCGAGTGCTATTTCACCAACGGGACGGAGCGGGTG CGGCTTCTGGCGAGAGACAT

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA

>dqb-lk-ewC

GATTTCGTGTTC

CTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGT CACGGAGCTCGGGCGGCCCg acGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGi:

TACGGGTTGGAAGAGCTCTACACGTTGCAGCGGCGA

>dqb-lk-ew88

GATTTCGTGTtCC

CTATAACCGGGAGGAGTACGTGCGCTTCGACAGCGACGTGGGGGAGTtCCGGGCGGT CACGGAGCTCGGGCGGCCCg acGCTGAGTACTGGAACCCGCAGAAGGACGAGATGGU

TACGGGTTGGAAGAGCTCtaCACGTTGCAGCGGCGA

>dqb-lk-023v

TACGGGTTGGAAGAGCTCACCACGTTGCAGCGGCGA

DLA-DRB

>DRBl*00101

CGTCGCTGAGTCCTGGAACGGGCAGAAGGAGATCTTGGAGCAGGAGCGGGCAACGGT GGACACCTACTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*00102

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00201

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*00202

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00301

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00401

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00501

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00601

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00701

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00801

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*00802

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*00901

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*010011

ACTACGGGGTGATTGAGAGCTTCaCGGTGCAGCGGCGAG

>DRBl*010012

CACATTTCGTGTACCAGTTTAAGCCCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTTCGTGGAAAGATAC

ATCCACAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCG GTCACGGAGCTCGGGCGGCC

ACTACGGGGTGATTGAGAGCTTCRCGGTGCAGCGGCGAG >DRBl*01101

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O12O1

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01301

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01302

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O14O1

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01501

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01502

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*01503

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01504

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01S01

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O17O1

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01702

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGCTCTTGGAGCGGGCGCGGGCCGCGGT GGACACCTACTGCAGACACA ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*01801

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*01901

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*02001

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*02101

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O22O1

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*02301

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*02401

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O25O1

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*02601

CACATTTCTTGC

ATCCATAACCGGGAGGAGAACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCG GTCACGGAGCTCGGGCGGCC

CGACGCTGAGTACTGGAACCGGCAGAAGGAGCTCTTGGAG

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*02701

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*02801

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*02901

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG

>DRBl*03001

CACATTTCTTGGAGATGGTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGCTTCTGGTGAGAGAC

ATCTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCG GTCACGGAGCTCGGGCGGCC

ACTACGGGGTGATTGAGAGCTTCRCGGTGCAGCGGCGAG

>DRBl*03101

CACATTTCTTGAAGATGGTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTATCTGATGAGAGAC

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*03201

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O32O2

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*03301

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O35O1

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*03601

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*03701

CACATTTCTTGgAGgTGGcAAAGgcCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTtcgTGgaaAGAtAC

CGACGCTGAGTCCTGGAACccGCAGAAGGAGCTCTTGGAGCgGgcGCGGGCCGCGGT GGACACCTACTGCAGACACA

ACTACGGGGTGggcGAGAGCTTCaCGGTGCAGCGGCGAG

>DRBl*03801

CACATTTCTTGGAGATGgTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGCtTCTGgTGAGAGAC

CGACGCTGAGTaCTGGAACGGGCAGAAGGAGCTCTTGGAGCgGAgGCGGGCCGaGGT GGACACggtgTGCAGACACA

ACTACcGGGTGATTGAGAGcTTCaCGGTGCAGCGGCGAG

>DRBl*04001

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRB1*O41O1

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*04201

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG

>DRBl*04301

CACATTTCTTGgAgAtGTTAAAGTTCGAGTGCCaTTTCACCAACGGGACGGAGCGGG TGCGGTATCTGGTGAGAGAC

ATCTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACG

CGACGCTGAGTACTGGAACCGGCAGAAGGAGCTCTTGGAG

ACTACCGGGTGGGCGAgAGCTTCACGGTGCAGCGGCGAG

JO

>DRBl*04401 CACATTTCTTGgAGgTGGcAAAGTcCGAGTGCtATTTCACCAACGGGACGGAGCGGGTGC GGTtagTGgaaAGAtAC

CGACGCTGAGTCCTGGAACcGGCAGAAGGAGCTCTTGGAGCAGAgGCGGGCCGCGGT GGACACCTACTGCAGACACA

ACTACcGGGTGggcGAGAGCTTCaCGGTGCAGCGGCGAG

>DRBl*04501

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O45O2

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O46O1

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*04701

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*04801

CACATTTCTTGGAGATGtTAAAGTcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGTtcgTGgaaAGAtAC

ATCcATAACCGGGAGGAGAaCgTGCGCTTCGACAGCGACGTGGGGGAGTaCCGGGCG GTCACGGAGCTCGGGCGGCC

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGCTCTTGGAGCgGAAGCGGGCCGaGGT GGACACCTACTGCAGACACA

ACTACgGGGTGattGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*04901

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*05001

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >DRBl*05101

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O52O1

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*05301

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*05401

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*05501

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*05601

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O57O1

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*05801

CACATTTCGTGTACCAGTTTAAGCCCGAGTGCCATTTCACCAACGGGACG

GAGCGGGTGCGGTTCGTGGAAAGATACATCCATAACCGGGAGGAGATCCT

GCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGTCACGGAGCTCGGGC

GGCCCGTCGCTGAGTCCTGGAACGGGCAGAAGGAGATCTTGGAGCAGGAG

CGGGCAACGGTGGACACGGTGTGCAGACACAACTACGGGGTGATTGAGAG

>drbl*05901

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >drbl*06101

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >drbl*06201

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*06301

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O64O1

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRBl*06501

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >DRB1*O66O1

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>DRBl*06701

CACATTTCTTGC

ATCTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCG GTCACGGAGCTCGGGCGGCC

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >jmadrb-ccahOO2

CACATTTCTTGGAGATGGTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGCTTCTGGTGAGAGAC ATCTATAACCGGGAGGAGCACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCGGTC ACGGAGCTCGGGCGGCC

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >jmadrb-dOO2

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >jmadrb~d004

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >jmadrb-vgl002

ACTACGGGGTGATTGAGAGCTTCGCGGTGCAGCGGCGAG >jsdrb-coyl057a

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >jsdrb-efinδder

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>j sdrb-hlatl7der

CACATTTCTTGAAGATC

ATCTATAACCGGGAGG;

CTCGGCTGAGTCCTGGAACCGGCAGAAGGAGTTCTTGGAGCAGAGGCGGGCCGAGGT GGACACGGTGTGCAGACACA

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>j sdrb-oest4der

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >j sdrb-ploolder

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >j sdrb-gfinllder

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >j sdrb-rest6der

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >lk03102

ACTACGGGGTGATTGAGAGCTTCaCGGTGCAGCGGCGAG >lkO35v-mw-u

ATCTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCG GTCACGGAGCTCGGGCGGCc

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGTTCTTGGAG

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-383 - 6

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >lkdrb-383-8

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-384-34

CACATTTCTTGAAGATGGTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTTTGTGGAAAGATAC

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG >lkdrb-awdθl

ACTACGGGGTGGGCGAGAgCTTCACGGTGCAgCGGCGAg >lkdrb-awdO2

ACTACGGGGTGattGAGAgCTTCACGGTGCAgCGGCGAg

>lkdrb-awdO3

CACATTTCgTGtACcaGtttAAGggCGAGTGCTATTTCACCAACGGGACGGAGCGGG TGCGGcTtcTGGcgAGAagC

CGACGCTGAGTACtgGAACCGGCAGAAGGAGcTCTTGGAGCAGagGCGGGCCGCGGT GGACACcTACTGCAGACACA

ACTACGGGGTGattGAGAgCTTCACGGTGCAgCGGCGAg

>lkdrb-awdO4

ACTACGGGGTGATTGAGAgCTTCACGGTGCAgCGGCGAg

>lkdrb-coy-r

CACATTTCTTGGAGGTGGCAAAGtyCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTTCGTGGAAAGATAC

CGACGCTGAGTCCTGGAACgGGCAGAAGGAGcTCTTGGAGCAGGAGCGGGCcgCGGT GGACACctacTGCAGACACA

ACTACcGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-coy-v

CACATTTCTTGGAGATGTtAAAGTtCGAGTGCcATTTCACCAACGGGACGGAGCGGG TGCGGTatcTGGtgAGAgAC

ATCtATAACCGGGAGGAGcACGTGCGCTTCGACAGCGACG

CGACGCTGAGTaCTGGAACGGGCAGAAGGAGCTCTTGGAG

ACTACGGGGTGattGAGAGCTTCgCGGTGCAGCGGCGAG

>lkdrb-coy-x

CACATTTCTTGGAGGTGGCAAAGgyCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTTCGTGGAAAGATAC

CGACGCTGAGTCCTGGAACcGGCAGAAGGAGaTCTTGGAGCAGGAGCGGGCaaCGGT GGACACggtgTGCAGACACA

ACTACgGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-015v-cl3

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >lkdrb-01802

CGGCGCTGAGTCCTGGAACCGGCAGAAGGAGCTCTTGGAGCGGAAGCGGGCCGAGGT GGACACGGTGTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-048v

CACATTTCTTGGAGATGtTAAAGTcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGTtcgTGgaaAGAtAC

ATCcATAACCGGGAGGAGcaCgTGCGCTTCGACAGCGACGTGGGGGAGTaCCGGGCG GTCACGGAGCTCGGGCGGCC

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGCTCTTGGAGCgGAAGCGGGCCGaGGT GGACACCTACTGCAGACACA

ACTACgGGGTGattGAGAGCTTCgCGGTGCAGCGGCGAG

>lkdrb-2332

CACATTTCTTGGAGaTGGtAAAGttCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTatcTGGAAAGATAC

CatCGCTGAGTcCTGGAACCgGCAGAAGGAGCTCTTGGAGCaGagGCGGGCCGCGGT GGACACCTACTGCAGACACA

ACTACGGGGTGattGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-5078

CACATTTCTTGGAgATGTTAAAGTtcgAgTGCCATtTCAcCAAcggGacggaGCGGG TGCGGTTCGTGGAAAGATAC

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGCTCTTGGAGCAGGAGCGGGCCGCGGT GGACACGGTGTGCAGACACA

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-9050

CACATTTCTTGGAGaTGGtAAAGTtCGAGTGCcATTTCACCAACGGGACGGAGCGGG TGCGGcTtcTGGtgAGAgAC

CGaCGCTGAGTaCTGGAACGGGCAGAAGGAGATCTTGGAGCAGGAGCGGGCAACGGT GGACACCTACTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-a79

CACATTTCGTGAAGATGTTTAAGGCCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGcTTCTGGCGAGAgaC

ATCTATAACCGGGAGGAGTTCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCG GTCACGGAGCTCGGGCGGCG

ACTACcGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-D7v

CACATTTCTTGGAGATGGTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTATgTGCTGAGAGAC

ATCTATAACCGGGAGGAGATCgTGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCG GTCACGGAGCTCGGGCGGCC

ACTACCGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-E17

CACATTTCgTGtAccaGtttAAGcCCGAGTGCcATTTCACCAACGGGACGGAGCGGG TGCGGTTCGTGGAAAGATAC

CGTCGCTGAGTCCTGGAACGGGCAGAAGGAGcTCTTGGAGCAGGAGCGGGCcgCGGT GGACACCTACTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-E25

CACATTTCgTGaAGaTGGCtAAGgCCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTTtCTGGcAAGAaAC

CGaCGCTGAGTCCTGGAACcGGCAGAAGGAGcTCTTGGAGCgGGAGCGGGCcgCGGT GGACACCTACTGCAGACACA

ACTACcGGGTGggcGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-E7

CGaCGCTGAGTCCTGGAACcGGCAGAAGGAGcTCTTGGAGCgGaAGCGGGCcgaGGT GGACACggtgTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-E25-2nd

CACATTTCgTGaAGaTGt11AAGtCCGAGTGCcATTTCACCAACGGGACGGAGCGGG TGCGGTatcTGGcgAGAgAC

CGaCGCTGAGTCCTGGAACcGGCAGAAGGAGcTCTTGGAGCgGGcGCGGGCcgCGGT GGACACCTACTGCAGACACA

ACTACcGGGTGggcGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-gw-c

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >lkdrb-gw-n

ACTACGGGGTGGGCGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-307

CACATTTCTTGaAGATGtcAAAGTcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGttggTGGaaAGAtgC

CtcgGCTGAGTcCTGGAACGGGCAGAAGGAGtTCTTGGAGCAGAaGCGGGCCGaGGT GGACACggtgTGCAGACACA ACTACGGGGTGggcGAGAGCTTCaCGGTGCAGCGGCGAG

>lkdrb-048v2

CACATTTCTTGGAGATGtTAAAGTcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGTtcgTGgaaAGAtAC

ATCcATAACCGGGAGGAGcaCgTGCGCTTCGACAGCGACGTGGGGGAGTaCCGGGCG GTCACGGAGCTCGGGCGGCC

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGCTCTTGGAGCgGAAGCGGGCCGaGGT GGACACCTACTGCAGACACA

ACTACgGGGTGattGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-7573

ATCtATAACCGGGAGGAGTaCGTGCGCTTCGACAGCGACGTGGGGGAGTACCGGGCG GTCACGGAGCTCGGGCGGCg

CGaCGCTGAGTCCTGGAACcGGCAGAAGGAGcTCTTGGAGCgGaAGCGGGCcgCGGT GGACACCTACTGCAGACACA

ACTACcGGGTGggcGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-7669

CGACGCTGAGTCCTGGAACCGGCAGAAGGAGcTCTTGGAGCGGAAGCGGGCCGaGGT GGACACggtgTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrb-3166

CACATTTCGTGAGGATGTATAAGGCCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGTaTCTGatGAGAgaC

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG >lkdrb3180

CtcgGCTGAGTCCTGGAACgGGCAGAAGGAGaTCTTGGAGCaGgAGCGGGCaacGGT GGACACCTACTGCAGACACA

ACTACGGGGTGATTGAGAGCTTCACGGTGCAGCGGCGAG

>lkdrbper475

CACATTTCTTGaAGATGGTAAAGTTCGAGTGCCATTTCACCAACGGGACGGAGCGGG TGCGGtTggTGGaaAGAGAC

CtcgGCTGAGTcCTGGAACcGGCAGAAGGAGtTCTTGGAGCAGAGGCGGGCCGcGGT GGACACctacTGCAGACACA

ACTACGGGGTGggcGAGAGCTTCaCGGTGCAGCGGCGAG

>drb-lk-ew31

ACTACGGGGTGATTGAGAGcTTCACGGTGCAGcggcgag

>drb-lk-ew56b

CACATTTCtTGgAggtGgcaAAGtcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGTTcgTGGaaAGAtaC

CgaCGCTGAGTaCTGGAACgGGCAGAAGGAGcTCTTGGAGCaGAaGCGGGCCGcGGT GGACACCTACTGCAGACACA

ACTACGGGGTGggcGAGAGcTTCACGGTGCAGcggcgag

>drb-lk-ew73b

CACATTTCGTGaggatGTTTAAGGcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGTTggTGGaaAGAgaC

CgaCGCTGAGTaCTGGAACgGGCAGAAGGAGcTCTTGGAGCaGAGGCGGGCCGAGGT GGACACCTACTGCAGACACA

ACTACcGGGTGggcGAGAGcTTCACGGTGCAGcggcgag

>drb-lk-ew88b

CACATTTCgTGaggatGTTTAAGGcCGAGTGCtATTTCACCAACGGGACGGAGCGGG TGCGGTTggTGGaaAGAgaC

ATCTATAACCGGGAGGAGTaCGTGCGCTTCGACAGCGACG

CATCGCTGAGTCCTGGAACCGGCAGAAGGAGTTCTTGGAG

ACTACcGGGTGggcGAGAGcTTCACGGTGCAGcggcgag

>drb-lk-8187

ACTACGGGGTGATTGAGAGcTTCGCGGTGCAGcGGCgAg

Identification of SNPs

In order to identity single nucleotide polymorphisms (SNPs) that would uniquely identify each MHC allele, the sequences of all the alleles set out above were compared to each other. The results of the comparison are shown in Tables 1 to 3. The names of each allele are shown in the left hand column. The numbers along the top row indicate the position in the sequence where each polymorphism occurs.

Table 1 - Comparison of DQA alleles

Table 2 - Comparison of DQB alleles

186 191

249 250

Table 3 - Comparison of DRB alleles

199 203

205206207209217218219220225236242243244254

Based on the comparison between the alleles, it is possible to specify a minimum number of SNP positions that need to be determined in order to identify a particular allele. These are set out in Tables 4 to 6.

Table 5 - DQB alleles

Table 6 - DRB alleles