# Patent

## Title
Screening for the genetic defect causing tibial hemimelia in bovines

## Abstract

The invention provides methods, materials and kits for analyzing DNA samples from bovine to determine whether the animal is a recessive carrier of a genetic mutation that is associated with tibial hemimelia. DNA-containing samples are analyzed by genetic testing to determine whether or not a deletion mutation is present in one of the alleles that encodes Aristaless-like4 (ALX4) protein, wherein the deletion mutation is associated with tibial hemimelia.

## Background
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was at least in part made with government support under AG 2004-34480-14417 and 58-5438-2-313 awarded by the USDA. The government has certain rights in the invention.


REFERENCE TO A SEQUENCE LISTING
A sequence listing containing SEQ ID NOs:1-103 is submitted as a text file with filename: 163-06 replacement seqlist.txt, created Oct. 18, 2011, 398841 bytes in size, and is specifically incorporated by reference.
BACKGROUND OF THE INVENTION
Tibial Hemimelia (TH) is a lethal congenital disorder in cattle characterized by severe and lethal deformities in newborn calves, including multiple skeletal deformities such as twisted rear legs with fused joints, large abdominal hernias and/or skull deformities. Often, such a calf is born dead, or if it survives birth cannot stand to nurse and must be destroyed, resulting in economic loss for owners.
TH was first described in Galloway cattle in the 1960's and in Shorthorn cattle in 2000. Since that time there have been hundreds of calves identified with TH. Extensive breeding and pedigree studies (see Marron et al. 2005; Ojo et al., 1974; Lapointe et al. 2000) have revealed that TH has an autosomal recessive mode of inheritance. For the TH phenotype to be expressed, a calf must have inherited the defective gene from both parents. A calf that expresses the phenotype is “homozygous” for the mutant TH gene, and the parents of such a calf are “heterozygous carriers” for the mutant TH gene. It is virtually impossible in the absence of planned breeding studies or test matings to classify whether a normal appearing individual is a heterozygous carrier of the mutant TH gene or is homozygous for the normal allele. Genetic screening is beneficial in avoiding loss of genetic resources due to culling based only on pedigree.
Because heterozygous individuals appear normal, carriers of the trait cannot be identified by eye, and instead exhaustive and time-consuming familial analysis must be done in order to identify potential carrier individuals. There is a need in the art for screens that can identify heterozygous carriers of TH by genetic testing to facilitate a breeding program that eliminates the genetic defect from the population. Such a screen requires an understanding of the genetic basis of the defect, including identification of the causative mutation within the DNA sequence. The present invention discloses a mutation associated with TH and provides a genetic test to determine whether apparently normal individuals carry a defective gene associated with TH. The present invention enables testing of individuals to determine whether an individual is a carrier. Individuals that are carriers can be removed from the breeding population, thereby facilitating removal of this genetic defect from the population.
While dramatic culling of suspected carriers would reduce the frequency of the mutation responsible for TH, such culling is long, expensive and can result in unnecessary reduction of beneficial genetic traits, as many of the culled animals would not be carriers of the mutation. Accordingly, there is a need in the art for a diagnostic or genetic screening test to determine whether or not an animal is a carrier of the mutation responsible for TH. The present invention provides materials and methods for screening animals to determine whether an animal is a heterozygous carrier of the mutant allele responsible for TH.
SUMMARY OF THE INVENTION
The invention features screens, methods, kits and associated probes, primers and DNA sequences for diagnosing in an animal, the genetic defect responsible for TH. The methods of the present invention are used to diagnose whether a phenotypically “normal” animal is a recessive carrier of a mutated gene which is associated with TH. In an embodiment, the method is for detecting a genetic defect in bovine genome that affects the ALX4 gene and, more particularly, a genetic defect that comprises a deletion mutation. The methods described herein are useful in detecting a deletion mutation greater than about 10,000 base pairs in length in the bovine genome that results in loss of ALX4 gene function. The specifically disclosed deletion is within SEQ ID NO:2, wherein about 45,693 base pairs are deleted, as reflected in SEQ ID NO:2 (wildtype gene), SEQ ID NO:3 (mutant TH gene) and SEQ ID NO:4 (deleted portion).
The methods of the invention rely on the finding that the mutation associated with TH is a deletion mutation. The deleted portion of the DNA corresponds to a “middle region” of the DNA sequence. The adjacent sequence portions upstream and downstream of this middle region correspond to an “upstream region” and “downstream region”, respectively. A normal TH genome (e.g., “non-mutant” or “wildtype”) has upstream, middle and downstream regions in a contiguous configuration. A mutant TH genome has at least one allele comprising the corresponding upstream and downstream regions in a contiguous configuration (e.g., the middle region is absent). In accordance with the present invention, each strand of a wildtype DNA molecule to be tested or screened comprises three regions: (i) an upstream region; (ii) a downstream region, and (iii) a sequence between the upstream and downstream regions. In a mutant DNA molecule associated with TH, the sequence between the upstream and downstream regions is deleted.
Accordingly, diagnostic assays and DNA tests of the present invention determine whether or not a deletion mutation within the region of the genome that encodes ALX4 is present. In an embodiment, the bovine genome comprises the bovine DNA sequence of SEQ ID NO:2 (wildtype) and/or SEQ ID NO:3 (mutant TH—found in TH-expressing phenotype and heterozygous TH carriers), wherein the middle portion corresponds to bases 29,693 to 75,385 of SEQ ID NO:2; also provided in SEQ ID NO:4.
An example of an upstream region DNA sequence is at least portion of the sequence upstream of, and contiguous to, base 29,693 of SEQ ID NO:2, or bases 1 to 29,692 of SEQ ID NO:2, or upstream, including and contiguous to base 1270 of SEQ ID NO:3, or bases 1-1270 of SEQ ID NO:3.
An example of a downstream region DNA sequence is at least portion of the sequence downstream of, and contiguous to, base 75,385 of SEQ ID NO:2, or bases 75,386 to 85,941 of SEQ ID NO:2, or downstream, including and contiguous to base 1271 of SEQ ID NO:3, or bases 1271 to 1686 of SEQ ID NO:4.
The DNA analysis optionally comprises PCR to amplify specific DNA sequences, thereby providing for accurate and reliable diagnostic and screening methods of the present invention. Primers are selected that flank regions of interest, including potential breakpoints or portions of the DNA sequence corresponding to the middle region. In an embodiment, a forward primer is selected that is capable of specific binding to the upstream region and a reverse primer is selected that is capable of specific binding to the downstream region. Such a primer pair cannot amplify DNA if the middle about 46 kb portion of DNA is present (e.g., wildtype), because the primers are sufficiently separated that amplification cannot efficiently occur. In the presence of the specifically exemplified deletion mutation, however, the two primers are close enough, for example less than about 5,000 base pairs, or less than 1,000 base pairs, or less than about 500 base pairs, for efficient amplification. The amplified DNA product is detected by any means known in the art, including with a probe (radioactive, fluorescent, luminescent or colored, for example), by a DNA sequencer, or by running the sample on an electrophoretic gel and detecting the DNA of an expected size.
Forward and reverse primers (as well as probes) useful in the present invention are shown in Table 3 in bold and in bold and underline. The probes and primers of the present invention comprise those having sequences corresponding to, or a reverse complement of, the bold or the bold and underlined sequences outlined in Table 3. Reverse primers correspond to reverse complementary sequences of the DNA sequences in bold or in bold underline. The invention includes the reverse complement sequences to obtain primer and probe sequences that specifically bind to targets of SEQ ID NOs:2-4, including specific targets within the upstream, middle, downstream, or potential breakpoint regions. A reverse primer is paired with a forward primer having a sequence with a region identical to at least a portion of the DNA sequences of any of SEQ ID NOs:2-4, including a region identical to at least a portion of the upstream region DNA sequence. Each of the indicated primers are capable of specific binding in that they do not span a DNA repeating sequence and do not have significant homology with any other DNA sequence of similar length. For example, the probes or primers may have up to seven adjacent nucleotides in common and have approximately 70% homology, including 70% and greater, with the corresponding target sequence given by a portion of any of SEQ ID NOs:2-4, or reverse complement thereof. Accordingly, the probes and primers are not limited to those explicitly exemplified, but encompass other probes and primers that one of ordinary skill in the art identifies as capable of specific binding. In addition, probes and primers specific to the breakpoint regions, (shown by the triangle in Table 4 and corresponding to between bases 1270 and 1271 of SEQ ID NO:4; upstream breakpoint between bases 29,692 and 29,693 of SEQ ID NO:2; downstream breakpoint between bases 75,385 and 75,386 of SEQ ID NO:2) are particularly useful in DNA-based analysis for determining TH deletion mutation status.
The two primer system that distinguishes between a (deleted) mutant gene and a normal gene relies simply on the presence or absence of an amplified DNA product, for example. If the primer pair flanks the potential deletion region, an amplified DNA product only occurs for the mutant and the animal is classified as “normal” if there is no amplified DNA product. Similarly, if the primer pair spans the breakpoint region (e.g., one primer specifically binds only if upstream and middle (or middle and downstream) is contiguous, absence of amplified product indicates the animal has a mutant allele. Accordingly, for quality control, addition of a third primer to the forward and reverse primers is desirable so that two distinguishable DNA products are generated which can then be distinguished by, for example, size or differentially-labeled probes. Preferably, the third primer is capable of specific binding to the middle region (or at least one end of the primer specifically binds to the upstream region and the other end specifically binds to the middle region, or alternatively one end of the primer binds to the downstream region and the other primer end binds to the middle region) so that a DNA product corresponding to the third primer and the forward primer is amplified. In this manner, every sample processed will have an amplified DNA product, which can be differentially detected and which serves as an internal control on PCR. Such a three-or-more primer system addresses a concern about whether lack of signal can be attributed to deficient PCR processing of an individual sample instead of whether or not there is a TH mutation.
In a particular embodiment, the DNA analysis further comprises providing one or more of a forward primer having the sequence of SEQ ID NO:5 (to specifically hybridize to the complementary strand corresponding to bases 29,619 to 29,640 of SEQ ID NO:2 or to bases 1,197 to 1,218 of SEQ ID NO:3), a reverse primer having the sequence of SEQ ID NO:6 (to specifically hybridize to bases 75,709 to 75,730 of SEQ ID NO:2 or to bases 1594 to 1615 of SEQ ID NO:3) and a third primer having the sequence of SEQ ID NO:7 (to specifically hybridize to bases 29,941 to 26,962 of SEQ ID NO:2; bases 249 to 270 of SEQ ID NO:4).
The DNA can be analyzed directly by providing a DNA probe that is capable of specific binding to a region that identifies the DNA as normal (e.g., the middle region, or the contiguous ends of the middle and upstream or middle and downstream regions) or is capable of binding to a region that identifies the DNA as a mutant (e.g., the breakpoint region). Alternatively, the DNA can be analyzed by DNA sequencing and comparing the sequences to those provided herein to determine whether there is a mutation. These techniques are optionally combined with PCR to generate an improved signal or additional information.
The oligonucleotide probes or primers of the present invention can be used with any of the methods disclosed herein. Probe or primer sequences are designed based on the DNA sequences provided herein, and specifically hybridize or bind to DNA regions so as to provide information about whether or not a deletion mutation in the ALX4 gene is present. In an embodiment, the probes or primers comprise a purified oligonucleotide having a length of about 15 to about 50 nucleotides. Particular specific binding sites include those encompassing bases 1270 and 1271 of SEQ ID NO:3, by the middle region defined by SEQ ID NO:4, and the middle region of bases 29,693 to 75,385 of SEQ ID NO:2 and contiguously associated upstream and downstream flanking regions.
The methods and materials provided herein can be used on any animal, and is preferably used in bovine to detect the presence or absence of a TH mutation, and is particularly useful in testing animals having any Shorthorn ancestry, for example Shorthorn and their composites. The tests and materials can be used with the DNA obtained from any animal tissue or fluid. Convenient samples are obtained from hair, blood or semen.
The invention encompasses an isolated and purified nucleic acid molecule of any of the sequences disclosed herein (e.g., those of Tables 3 and 4, and any of the SEQ ID NOs), or including at least a functional fragment thereof. Useful primers and probes include those that specifically bind a target sequence that resides in at least a portion of a particular DNA region such as an upstream region, downstream region or a middle region. Other useful oligonucleotide primers or probes include those that specifically bind a breakpoint or those that specifically bind two adjacent regions, such as an isolated and purified nucleic acid molecule comprising at least a functional fragment of a deletion breakpoint that is the causative agent of TH. The probes or primers can be of any length and homology, so long as the length and homology is sufficient to result in specific binding to a specified target region. In an embodiment, the probe or primer is an oligonucleotide or a DNA sequence that ranges in size from about 15 to 80 bases, or about 18 to 60 bases, or about 20 to 25 bases. Desirably, at least 12, at least 15, or preferably at least 20 bases are homologous to the target sequence. In an embodiment, all the primer or probe base are homologous (e.g., complementary) to the target sequence. Exemplary probe or primer sequences are provided in Table 3 in bold, and also in bold and underline.
Kits comprising any of the oligonucleotide probes or primers of the present invention are within the scope of the invention. The kits can further comprise instructions for appropriate DNA processing, hybridization and/or PCR conditions, and for visualizing or detecting amplified DNA products.
For quality control, the kit optionally comprises DNA test samples that are a positive control (e.g., a mutant DNA sample comprising the breakpoint indicated in Table 4; corresponding to between bases 1270 and 1271 of SEQ ID NO:3) and/or test samples that are a negative control comprising the middle region and associated flanking upstream and downstream regions, as summarized for SEQ ID NO:2. These controls can comprise DNA sequences corresponding to expected DNA-amplified products from the primers of the kit, or can be isolated and purified DNA sequences corresponding to wildtype or to normal that can be used by the probes or primers of the kit.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows two pedigrees (A and B) representing a panel of 61 animals used for mapping the TH locus. Bulls are represented by squares and cows are represented by circles. Hatched circles or squares refer to carrier animals and filled diamonds refer to affected calves (e.g., TH phenotype expressed). Haplotypes are located beneath each animal on the panel. Filled chromosomes represent the TH haplotype. Hatched alleles and those in parenthesis are inferred.
FIG. 2 is an image of the results of a DNA-based test for tibial hemimelia (TH). PCR amplification using primers capable of simultaneous amplification of a normal chromosome segment and a mutated chromosomal segment is used to determine TH status in each of ten DNA samples from different animals. Animals in lanes 1, 6 and 9 are homozygous normal due to the presence of only the DNA segment representing the normal chromosome. Animals in lanes 2, 4 and 8 are homozygous for the chromosome with the deletion mutation causing TH, indicating that the samples were taken from affected calves. Animals in lanes 3, 5, 7 and 10 possess both DNA segments, indicating they are heterozygous and carriers of the mutation associated with TH.



DETAILED DESCRIPTION OF THE INVENTION
TH is observed in Shorthorn cattle and Shorthorn influence cattle, including but not limited to non-purebred registered Maine-Anjou, Chianina, Santa Gertrudis and Simmental and any crossbreds. In particular, any animal that can be traced back to a particular Shorthorn bull imported to the US in the 1970's is potentially a carrier of the defective gene responsible for TH. Analysis of lineage lines indicates that TH is an autosomal recessive disease. Accordingly, many animals in the breeding population are potential “heterozygous carriers”, e.g., animals that have one copy of the gene responsible for TH. This number is estimated to be as high as 10% in certain Shorthorn breeds. The methods and compositions of matter presented and claimed herein are particularly useful for diagnosing whether or not an animal is a recessive carrier of the defective gene responsible for TH. These animals can be removed from the breeding population so as to breed out the gene responsible for TH.
As used herein, “DNA sample” includes the part of the bovine genome that is a locus for TH. In particular, it is that part of the genome associated with expression of ALX4. The invention, accordingly, is useful for detecting whether or not there is a deletion mutation that when inherited from both parents results in phenotypic expression of TH. An animal that is a heterozygous carrier of this deletion mutation is said to have a mutation that is associated with TH.
“Obtaining” is used broadly to refer to any method of obtaining a biological sample that contains DNA, and specifically a sample that contains at least a portion of the genome spanning a region associated with the mutation that is a causative agent of TH. The sample can be from any tissue, so long as the sample contains DNA that is not significantly degraded, and can be fresh, frozen or otherwise preserved. For example, blood, semen or hair are relatively easily obtained and can be processed for immediate analysis or stored for later transport, processing and analysis.
As used herein, “analyzing” broadly refers to any technique that reveals genetic information, particularly whether or not a DNA sample contains an allele comprising a mutation associated with TH. In a preferred embodiment, the technique comprises PCR processing to amplify selected DNA sequences to yield information about the status of the sample. Other techniques, including DNA hybridization with probes, DNA sequencing, DNA separation by gel electrophoresis and others known in the art can be optionally combined with PCR to generate improved signals.
The portion of the bovine genome that is involved with TH, including those portions useful for determining whether an animal is normal or a heterozygous carrier of TH, can be divided into three regions: (i) an upstream region; (ii) a downstream region; and (iii) a middle region, wherein the middle region forms a contiguous configuration with the upstream region at one end and the downstream region at the other end. “Contiguous configuration” refers to two or more DNA sequences forming a continuous DNA strand, wherein there are no additional DNA sequences between adjacent strands. The invention is based on the discovery that the TH mutation is associated with deletion of the middle region, such that the upstream region and downstream region form a contiguous configuration. DNA analysis of known TH mutant genes reveals that the mutant gene has a deletion, whose size and location tends to be conserved among different animals. With this information, methods and related compositions of matter are presented herein that are useful in determining whether or not an animal is a recessive carrier of a TH mutant gene by determining the presence or absence of this middle region.
Polymerase Chain Reaction (“PCR”) is a technique in which cycles of denaturation, annealing with primer, and extension with DNA polymerase are repeatedly used to amplify the number of copies of a DNA segment, up to and greater than 106 times. PCR and associated PCR conditions are known in the art and are described more fully in U.S. Pat. Nos. 4,683,195 and 4,683,292, which are herein incorporated by reference. A “primer” is a single stranded oligonucleotide or DNA fragment which hybridizes to a DNA strand. In PCR, primers are generally paired, with a 5′ forward primer that hybridizes with the 5′ end of the DNA sequence to be amplified, and a 3′ reverse primer which hybridizes with the complement of the 3′ end of the sequence to be amplified. The amplified DNA sequence encompasses the target sequence hybridized by both primes, as well as the intervening sequence between both primer target sequences. Any portion of the DNA sequences provided herein can be used as a probe or primer, so long as the probe or primer sequence specifically binds one target. Such specific binding improves the reliability of DNA-based screens useful for identifying carriers of the TH mutation.
The oligonucleotide primers and probes are generally selected for their ability to specifically bind to at least a portion of the upstream, downstream or middle DNA region. “At least a portion” refers to the embodiment where the target DNA sequence spans adjacent regions, including upstream-downstream (e.g., TH mutant), upstream-middle (e.g., no TH mutant) or middle-downstream regions (e.g., no TH mutant).
As used herein, “Shorthorn composite” refers to any bovine animal with any Shorthorn ancestry.
Analyzing DNA encompasses any means known in the art: cleavage, where cleavage is dependent on whether or not there is a deletion mutation; hybridizing of probes, where probe binding is dependent on whether or not there is a deletion mutation; PCR amplification, where the presence of amplification products, or the size of the amplification products, depend on whether the deletion mutation is present; DNA sequencing; etc. The invention can be practiced with any DNA detection methods known in the art, including any future-arising detection methods. Analysis methods rely on the discovery of a deletion mutation that is associated with TH, as reflected in the difference between the wildtype genetic sequence (SEQ ID NO:2) and the TH mutant genetic sequence (SEQ ID NO:3), and more specifically, the recognition that a large deletion mutation that is located within a defined location in the genome is associated with the disease (SEQ ID NO:4, corresponding to breakpoint between bases 1270 and 1271 of SEQ ID NO:3). Some examples of methodology that can be useful in detecting whether or not a large deletion is present include U.S. Pat. Nos. 4,683,202 (Process for Amplifying Nucleic Acid Sequences), 6,013,444, 6,225,093 US Pat. Pub No. 2006/0063191 (Detecting Nucleic Acid Deletion Sequences).
The typical DNA deletion of the TH gene is greater than about 40 kb (e.g., the difference between SEQ ID NO:2 and SEQ ID NO:3 is a 45,693 length deletion of SEQ ID NO:4). Accordingly, if this sequence is present (e.g., the genome is from wildtype), under typical PCR conditions (e.g., “short PCR conditions”), a primer pair located on either side of this deletion sequence will not generate any amplified product. If, however, the mutation is present and this sequence is in fact deleted from the DNA sample, the primer pair is then sufficiently close to result in generation of an amplified DNA product that is then detected.
In the use of the oligonucleotides or polynucleotides as probes, the particular probe is labeled with any suitable label known to those skilled in the art, including radioactive and non-radioactive labels. Typical radioactive labels include 32P, 35S, or the like. Non-radioactive labels include, for example, ligands such as biotin or thyroxine, as well as enzymes such as hydrolases or peroxidases, or a chemiluminescer such as luciferin, or fluorescent compounds like fluorescein and its derivatives. Alternatively, the probes can be made inherently fluorescent as described in International Application No. WO 93/16094.
Various degrees of stringency of hybridization can be employed. The present invention contemplates nucleic acid sequences which hybridize under low, moderate or high stringency hybridization conditions to the exemplified nucleic acid sequences set forth herein. Duplex formation and stability depend on substantial complementarity between the two strands of a hybrid, and a certain degree of mismatch can be tolerated. The more stringent the hybridization conditions, the greater the complementarity that is required for duplex formation. Stringency can be controlled by temperature, probe concentration, probe length, ionic strength, time, and the like. Preferably, hybridization is conducted under moderate to high stringency conditions by techniques well known in the art, as described, for example in Keller, G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y., pp. 169-170, hereby incorporated by reference. For example, stringent conditions are those that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate (SSC); 0.1% sodium lauryl sulfate (SDS) at 50° C., or (2) employ a denaturing agent such as formamide during hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C. Another example is use of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 times Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% sodium dodecylsulfate (SDS), and 10% dextran sulfate at 4° C., with washes at 42° C. in 0.2×SSC and 0.1% SDS.
An example of high stringency conditions is hybridizing at 68° C. in 5×SSC/5×Denhardt's solution/0.1% SDS, and washing in 0.2×SSC/0.1% SDS at room temperature. An example of conditions of moderate stringency is hybridizing at 68° C. in 5×SSC/5×Denhardt's solution/0.1% SDS and washing at 42° C. in 3×SSC. The parameters of temperature and salt concentration can be varied to achieve the desired level of sequence identity between probe and target nucleic acid. See, e.g., Sambrook et al. (1989) supra or Ausubel et al. (1995) Current Protocols in Molecular Biology, John Wiley & Sons, NY, N.Y., for further guidance on hybridization conditions.
In general, salt and/or temperature can be altered to change stringency. With a labeled DNA fragment >70 or so bases in length, the following conditions can be used: Low, 1 or 2×SSPE, room temperature; Low, 1 or 2×SSPE, 42° C.; Moderate, 0.2× or 1×SSPE, 65° C.; and High, 0.1×SSPE, 65° C.
“Complement” or “complementary sequence” means a sequence of nucleotides which forms a hydrogen-bonded duplex with another sequence of nucleotides according to Watson-Crick base-pairing rules. For example, the complementary base sequence for 5′-AAGGCT-3′ is 3′-TTCCGA-5′. This invention encompasses complementary sequences to any of the nucleotide sequences claimed in this invention.
A “functional fragment” of a nucleic acid is a partial sequence of the nucleic acid molecule such that the functional fragment has utility as a probe, primer or a target sequence for specific binding to a complementary probe or primer in the present invention, for example. A functional fragment that is a probe or a primer is useful for diagnosis, sequencing or cloning of the portion of the DNA genome that is associated with TH, including the portion of the genome that encodes ALX4, and that portion of the genome that comprises a deletion mutation associated with TH.
In accordance with the present invention, there is provided a purified and isolated nucleic acid molecule which regulates and encodes for ALX4 protein. Desirably, the nucleic acid molecule is a DNA isolated from Shorthorn or Shorthorn composites. Further encompassed are nucleotide sequences for probes and primers to various portions of the genome associated with the ALX4 gene, and in particular probes and primers that bind specifically to an upstream region, downstream region, or a middle region, wherein the middle region corresponds to a mutation deletion associated with TH. Given a particular sequence, the generation of primers to that sequence is well known in the art. Sequencing and diagnostic primers are typically 20 to 28 base pairs, more preferably 22 base pairs in length, and generally match the sequence of interest between approximately 90% to 100%, most preferably approximately 100%. Primers are typically approximately 20 to 34 base pairs in length, more preferably 21 to 24 base pairs in length, with annealing temperatures in the 50 to 70° C. range. Gene probes are preferably approximately 1 kb in length comprising the gene of interest to be probed.
Particular probe or primer sequences are selected for their ability to bind a single specific region of the DNA sequence in one or more of SEQ ID NOs:2-4, (e.g., “specific binding”), but not to other genomic loci. As used herein, “specific binding” or “binds specifically” refers to an oligonucleotide (e.g., a primer or probe) that is sufficiently selective in hybridizing the target sequence so as to result in DNA analysis that is reliable and accurate in identifying DNA having a TH mutant allele. A probe or primer that is capable of specific binding to a DNA target sequence does not hybridize in significant amounts (e.g., measurable) amounts to a non-target sequence. To ensure specific binding, a number of different considerations are employed. For example, none of the target sequences should be located in DNA repetitive elements. In addition, potential target sequences can be analyzed against the remainder of the sequence to determine whether there are other regions with significant homology. “Homology” or “sequence identity” means the proportion of base matches between two nucleic acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the fraction of matches over the length of sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching. When using oligonucleotides as probes, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and more preferably not less than 19 matches out of 20 possible base pair matches (95%). A primer or probe sequence of the present invention is considered capable of specific binding if there is less than 80% homology, less than 70%, and preferably less than 50% homology (corresponding, to a 20 base pair probe or primer having less than 16, less than 14, or less than 10 identically aligned bases) to other “non-target” sequences.
Hybridizing the probes or primers with the DNA sample under stringent conditions also reduces the likelihood of binding to regions other than the target region. In general, probes or primers having higher homology to other sequences besides the target sequence can be hybridized under more stringent conditions than probes or primers having lower homology.
For regular PCR conditions, the location of primer pairs are preferably separated by less than about 4,000 base pairs, less than about 2,000 base pairs, or less than about 500 base pairs, thereby ensuring efficient amplification. The invention is not, however, limited to a specific primer separation distance; the constraint is the ability of the primers to generate amplified DNA.
As used herein, the terms “isolated and/or purified” refer to in vitro isolation of a DNA molecule from its natural cellular environment and from association with other components of the cell, such as nucleic acid, so that it can be sequenced, replicated, amplified and/or expressed. An “isolated and purified nucleic acid molecule” is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid. This term covers, for example, DNA which has part of the sequence of a naturally occurring genomic DNA, but does not have the flanking portions of DNA found in the naturally occurring genome. The term also includes, for example, a nucleic acid incorporated in a vector or into the genome of a cell such that the resulting molecule is not identical to any naturally occurring vector or genomic DNA.
All references cited throughout this application, for example patent documents including issued or granted patents or equivalents; patent application publications; and non-patent literature documents or other source material are hereby incorporated by reference in their entireties, as though individually incorporated by reference, to the extent each reference is not inconsistent with the disclosure in this application (for example, a reference that is partially inconsistent is incorporated by reference except for the partially inconsistent portion of the reference).
Every formulation or combination of components described or exemplified herein can be used to practice the invention, unless otherwise stated.
Whenever a range is given in the specification, for example, a temperature range, a size range, a time range, or a composition or concentration range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure. It will be understood that any subranges or individual values in a range or subrange that are included in the description herein can be excluded from the claims herein.
All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference in their entirety to indicate the state of the art as of their publication or filing date and it is intended that this information can be employed herein, if needed, to exclude specific embodiments that are in the prior art. All tables attached hereto (e.g., Tables 1-6) are part of the specification.
As used herein, “comprising” is synonymous with “including,” “containing,” or “characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of” does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms “comprising”, “consisting essentially of” and “consisting of” may be replaced with either of the other two terms. The invention illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.
One of ordinary skill in the art will appreciate that starting materials, materials, reagents, synthetic methods, purification methods, analytical methods, assay methods, and methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such materials and methods are intended to be included in this invention. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
EXAMPLE 1
Mapping of the Locus Causing TH in Shorthorn Cattle
Tibial Hemimelia is a congenital abnormality present in Shorthorn cattle that is characterized by severe and lethal deformities in newborn calves. These deformities include malformed tibias, abdominal hernias, and meningoceles. Pedigree analysis indicates an autosomal recessive mode of inheritance with a single proband sire. DNA samples from 17 affected calves along with their sires and most dams are collected as well as samples from putative homozygous normal individuals, not related to the proband. A total of 264 microsatellite markers distributed evenly across bovine autosomes are selected for genotyping and linkage analysis. Genotypic data is analyzed for all chromosomes having candidate genes corresponding to human and mouse disease loci with similar phenotypes. This analysis revealed significant linkage between BTA15 and the disease locus. Results of this study provide a foundation for the discovery of the causative mutation of TH and the development of a DNA-based diagnostic test.
The tibial hemimelia (TH) genetic defect was first recognized in Shorthorn cattle in 2000 (Lapointe et al., 2000), but may have been recognized in Galloway cattle in the early 70's (Ojo et al., 1974). The defect is characterized by multiple skeletal deformities, most notably shortened or absent tibia. The tibial deformity can range from bilateral shortening or malformation of the tibia with joint fusion to a completely absent tibia. Other characteristics of the disease may include abdominal hernia, due to incomplete fusion of the pelvic symphysis, presence of meningocele, and a long, shaggy coat. Calves are born dead, or fail to thrive and die shortly after birth (Lapointe et al., 2000).
Pedigree analysis of the affected calves indicates a single common index sire within 6-7 generations. The defect appears in equal frequency between sexes and all parent individuals are normal, indicating an autosomal recessive mode of inheritance. Evidence from prior breeding studies in Galloway cattle suggests the involvement of only a single gene (Leipold et al., 1978).
Prior to the present invention, the frequency of the TH locus is unknown. The prevalent use of asymptomatic carrier animals in breeding populations has resulted in an increase of TH in Shorthorn populations. Currently, progeny testing is the only available testing method used to determine TH status of breeding animals. Therefore, a DNA-based test for the causal mutation is of great importance to identify carrier animals without the need to perform breeding trials. The first step towards this DNA-based test is the identification of the chromosomal location of the TH locus.
Possible candidate genes are selected based on similar phenotypes in humans and mice. Bovine chromosome segments corresponding to human and mouse regions containing these candidate genes are identified based on comparative mapping data (Everts-Van der Wind et al., 2004). Chromosome screening is prioritized based on the number of candidates located on each chromosome. A panel of 61 animals with known TH genotypes is used for mapping the TH locus. These animals are contained within 2 pedigrees and include 17 affected calves, 4 normal, and 40 carrier animals. Over 260 microsatellites from the USDA-MARC map, approximately 10-15 cM apart are selected for genotyping this panel. PCR is carried out in 10 μl reactions containing 1×PCR buffer, 200 μM each dNTP, 0.5 μM each primer, 0.25 U of HotstarTaq polymerase (Qiagen), 5 μCi of [α-32P] dCTP, and 30 ng of genomic DNA. Reactions are incubated at 95° C. for 15 minutes followed by 35 cycles of 94° C. for 30 seconds, 50-65° C. for 45 seconds, and 72° C. for 45 seconds, with a final incubation at 72° C. for 10 minutes. PCR products are analyzed on 7% acrylamide gels. Upon completion of each chromosome, linkage analysis is performed using CRI-MAP v. 2.4.
A total of 39 candidate genes are identified on 16 human chromosomes corresponding to 19 different bovine chromosomes. Among these, 2 chromosomes containing three or four candidate genes, 6 with two candidate genes, 9 with one candidate gene, and 10 with no candidate genes are identified. Genotyping is completed for 179 markers corresponding to chromosomes 1-8,10,14-16, 21 and 25. Following two-point linkage analysis, one chromosome (BTA15) exhibited strong linkage to the TH locus (FIG. 1). Additional markers are used to genotype the panel in order to fine map the region. The most significant LOD scores are exhibited between TH and BL1095 (5.85), DIK2382 (4.82), BMS820 (8.73), and IDVGA23 (8.73) (Table 1). Using information from affected calves, recombination events between the TH haplotype found in the proband sire and the normal haplotype are used to construct a critical region in this area of BTA15. The haplotype analysis suggests that the TH locus lies between markers 6 and 7 (BL1095 and BMS820) (Table 2).
A comparative genomics approach is used to map the TH locus. This approach is invaluable for both the identification of possible candidate genes as well as prioritization of data collection. Upon examination of homologous conditions in humans and mice, a list of candidate genes responsible for the TH phenotype is assembled. Using the human-bovine comparative map (Everts-Van der Wind, et al. 2004), cattle chromosomes of interest i.e. those containing candidate genes, are identified and analyzed quickly. The strong linkage between two markers on BTA15 and TH prompted genotyping of additional markers in that region. LOD scores for these markers revealed significant linkage to a region between BL1095 and IDVGA23, between 94.7 and 99.9 cM, or approximately a 5 cM region. In order to decrease this region, recombination events between affected calves and the proband sire are analyzed. The haplotype analysis revealed a critical region between BL1095 and BMS820 surrounding the marker DIK2382, corresponding to roughly 95-98 cM. Bovine microsatellite sequences surrounding the critical region are compared to the human genome by BLAST analysis. The microsatellites were homologous to the human sequence between 42.3 Mb-45.2 Mb on HSA 11. One candidate gene identified in the beginning of the study is located in this region of HSA11. The candidate gene in this region is Aristaless-like4, or ALX4 gene. This gene is picked as a candidate due to its strong role in limb formation. Previous studies have linked mutations in this gene to such deformities as parietal foramina (Wu et al., 2000 and Wuyts et al., 2000) and polydactyly (Qu et al., 1998 and 1997).
EXAMPLE 2
Location and Sequence of a Deletion Mutation Responsible for TH
The general location on the bovine genome of the mutation associated with TH allows focused examination of DNA sequences in that location so as to identify the genetic mutation. Table 3 (see SEQ ID NOs:1-2) provides the sequence for bovine EXT2 and ALX 4 genes from a bovine (Hereford used to generate the CHORI-240 library for the bovine genome sequencing project). The sequence in Table 3 corresponds to the bovine DNA sequence that encompasses the exostoses (multiple) 2 (EXT2) and aristaless-like homeobox 4 (ALX4) genes. Exons corresponding to the protein coding sequence of ALX4 are highlighted and labeled as ALX4_EXON1, ALX4_EXON2, ALX4_EXON3 and ALX4_EXON4. Investigation of normal and mutant alleles reveal that a mutation associated with TH comprises a deletion of the sequence highlighted in gray, running from BREAKPOINT A to BREAKPOINT B. Exon 1 of the ALX4 gene is within this deleted segment region along with associated regulatory sequences. SEQ ID NO:4 contains the sequence of this mutation deletion region. Various probes and/or primers useful in defining the precise breakpoint region as well as for diagnostic tests and screens are in bold or in bold underline.
The portion of the sequence in Table 3 with repeating “N” indicates a sequence gap that is less than about 2 kb. SEQ ID NO:1 corresponds to the sequence upstream of the gap. SEQ ID NO:2 is the sequence contained downstream of this gap, and includes the upstream, middle and downstream regions of the portion of the genome that regulates a protein (ALX4) that is associated with TH. The middle region comprises bases 29,693 to 75,385 of SEQ ID NO:2 and is provided in SEQ ID NO:4. The upstream region is at least a portion of the SEQ ID NO:2 that end with base 29,692. The downstream region is at least a portion of SEQ ID NO:2 wherein the sequence begins at base 75,386. The upstream and downstream regions of the presently claimed invention can be of any length, so long as it is possible for a probe or primer to specifically bind the region in a manner required to carry out the claimed invention.
The deletion breakpoint is determined by PCR amplification across the deletion breakpoint for DNA obtained from TH calves or cattle known to be heterozygous carriers of the TH mutation. Table 4 (corresponding to SEQ ID NO:3) is a sequence obtained from such PCR studies and an arrow highlighted in grey (“▴”) (corresponding to between bases 1270 and 1271 of SEQ ID NO:3) indicates the position of the deletion breakpoint. Referring to SEQ ID NO:3, this breakpoint deletion mutation is located between bases 1270 and 1271, such that an upstream region comprises a DNA segment ending at base 1270, and a downstream region comprising a DNA segment beginning at base 1271, wherein the upstream and downstream regions are in a contiguous configuration. As summarized by the shaded portion of Table 3 (corresponding to SEQ ID NO:4), the deleted portion encompasses the 5′ regulatory region and exon 1 of the ALX4 gene that controls early events in limb bud (e.g., hind legs) formation. This mutation results in complete loss-of-function of ALX4, thus producing the disease phenotype when an animal is homozygous for the deletion-containing chromosome. Corresponding gene “knock-out” models in mice result in similar pathologies.
BLAST analysis (Table 5) shows the alignment between the sequence of Table 3 (wildtype) and Table 4 (mutant), indicating that the mutant sequence comprises flanking sequences (e.g., upstream region and downstream region) of Table 3 with a middle region of 45,693 deleted.
SEQ ID NO:4 is the sequence of the entire middle region of the bovine genome, wherein the absence of this region is associated with a mutation responsible for TH.
Those of ordinary skill in the art will recognize that any number of DNA-based diagnostic tests can be developed that detect the presence or absence of a deletion on the order of greater than about 30,000 base pairs, or greater than about 45,000 base pairs, or about 45,693 base pairs. Given the length of the DNA sequence of SEQ ID NOs:2 and 4, the invention tolerates variation in both the precise breakpoint location and breakpoint sequence. For example, primers and probes can be designed so that the breakpoints (e.g., between bases 29,692-29,693 and bases 75,385-75,386) can vary, for example by plus or minus 20 base pairs, or plus or minus 10 base pairs, or plus or minus 5 base pairs or less, without affecting the ability of the probe or primer to specifically bind the target sequence and generate an output signal that can be used to determine the presence or absence of a deletion. One example of such designing is to ensure the probe/primer sequence is of adequate length, and also to target sequences that are greater than 5, greater than 10 or greater than 20 base pairs away from the potential breakpoint location.
EXAMPLE 3
DNA-Based Test for Tibial Hemimelia
Any DNA test that detects the breakpoint of the sequence that is associated with TH (e.g., between about bases 29,693 to 75,385 deleted from SEQ ID. NO:2, corresponding to the breakpoint between base G1270 and G1271 of SEQ ID NO:3) can be used as the basis of an assay for testing whether a subject is a carrier of the TH gene. Such tests include but are not limited to DNA sequencing, hybridization and allele-specific extension. For example, PCR can be used to amplify appropriate DNA portions, and the amplified DNA run on a gel that separates DNA by size. In this example, such a PCR test uses three different primer sequences, a first (forward) primer that binds upstream of the breakpoint, a second (reverse) primer that binds downstream of the breakpoint, and a third primer that specifically binds to the middle region, corresponding to the deleted portion of DNA associated with the mutation responsible for TH. The actual location of the target sequence to which the primer specifically binds is not critical, although under “normal” PCR conditions (e.g., not long range PCR conditions, see U.S. Pat. No. 6,225,093) it is preferable if the target sequence is within about 5 kb, or within 1 kb, or within about 500 bases of a potential breakpoint site and the other end of the to-be-amplified DNA sequence. In one embodiment, the primers used are as follows:







First (forward) primer (SEQ ID NO: 5):


TH_BIGBREAK_F (5′-TGCTCAGGCTGGTTTCTCTTCC-3′)


(corresponding to bases 29,619 to 29,640 of SEQ


ID NO: 2; bases 1,197 to 1,218 of SEQ ID NO: 3)


 


Second (reverse) primer (SEQ ID NO: 6):


TH_BIGBREAK_R (5′-GTGCAAAGACAAGGCCTCTCGT-3′)


(reverse complement of bases 75,709 to 75,730 of


SEQ ID NO: 2 or of bases 1594 to 1615 of SEQ ID


NO: 3).


 


Third (binding to middle region) primer (SEQ ID


NO: 7) TH_BIG_344C (5′-CACCCAGTATAGTCAGCAGCGT-3′)


(reverse complement of bases 29,941 to 26,962 of


SEQ ID NO: 2, or of bases 249 to 270 of SEQ ID NO: 


4; primer does not specifically bind to SEQ ID NO: 


3).






The names of the primers correspond to those provided in Table 3. This combination of primers is used under standard PCR conditions to generate the test results as depicted in FIG. 2. The first and second primers are each located on a separate side of the breakpoint. Accordingly, in a normal allele there is no amplification product attributed to the first and second primers as the intervening about 45 kb sequence between the first and second primers prevent generation of PCR-amplified DNA product. If there is, however, an allele for the TH disease (e.g., the intervening about 45 kb sequence is deleted), a first DNA product is amplified, wherein the DNA corresponds to the region encompassed by the first and second primers. The resultant length of this first DNA product is L12. The first and third primers generate a DNA amplified fragment corresponding to the normal chromosome having length L13. The third primer resides in the deleted portion, and so a DNA product is not amplified for a TH mutant chromosome. DNA of length L13 is generated only if the DNA sequence is wildtype. By selecting precise target sequences, the size of the amplification products generated by primers 1 and 3 (L13) can be different than the size generated by primers 1 and 2 (L12). The amplified DNA can be run on a gel to separate DNA by size, with the observed pattern dependent on whether or not the DNA from the subject to be tested is a carrier of a TH gene, as summarized in Table 6. FIG. 2 shows that two bands in a lane indicates a chromosome having one allele that has the mutation and another allele that is normal (lanes 3, 5, 7 and 10), thereby identifying the subject as a heterozygous carrier of the TH gene. If only one band is observed, the subject is either normal (lanes 1, 6 and 9) or the sample is from an individual that expresses the TH phenotype (e.g., both alleles have the TH mutation (lanes 2, 4, and 8)), and can be distinguished by size.
As summarized in Table 6, the third primer is not required in order to distinguish a normal animal from a heterozygous TH carrier. For quality control reasons, however, the third primer is optionally present and ensures that the DNA has been appropriately amplified and detected. As known in the art, the DNA need not be run on a gel, rather probes specific to each of the amplification products can be used, including radiolabeled, fluorescently labeled or any other detection substances and associated means for detecting the substance. Size separation and DNA labeling with, for example ethidium bromide is, however, a low-cost and easily performed assay for detecting TH heterozygous carriers.
In an aspect, the amplified DNA products are detected, thereby identifying heterozygous carriers of a gene that is associated with TH. In an embodiment the DNA product is detected by running the DNA on a size-separation gel, wherein the expected sizes of each DNA product is known. Isolated and purified DNA sequences of the present invention corresponding to (1) normal, and (2) mutant can be used to ensure PCR conditions and DNA analysis is functioning appropriately.
A unique, DNA-based diagnostic test that accurately determines the (TH) genotype status within Shorthorn cattle populations through analysis of DNA containing samples i.e. blood, semen or hair follicles is important for eliminating this genetic defect from the population. Such a test eliminates the need for parental validation. FIG. 2 is an example of one such test where PCR amplification of the DNA from each individual is used to determine TH status. This diagnostic assay is supported by three independent validation experiments, including blind analysis of 48 samples of known genotype status based on progeny analysis, analysis of phenotypically normal individuals of suspect pedigree, and analysis of unrelated cattle breeds for the presence of the mutation. Results of the blind sample analysis were 100% concordant with the known genotypic status of the individuals. Among all phenotypically normal individuals of suspect pedigree, as expected no individual was genotyped as homozygous for the deletion mutation. This is further support that that those animals that express the TH phenotype are homozygous for the deletion mutation. Ongoing experiments of bovine animals indicate the deletion mutation is present within Shorthorn and Shorthorn composite animals.
As understood in the art, genomic DNA comprises a sense strand and an antisense strand that is complementary to the sense strand. The sequences listed herein, are to the sense strand, running 5′ to 3′. Accordingly, the invention comprises the corresponding antisense sequences that are complementary to the listed sequences, and further include the reverse complementary sequences of all the primers and probes disclosed herein.
SEQ ID NO:1—DNA sequence upstream of gap region
SEQ ID NO:2—Wildtype DNA sequence comprising upstream, middle and downstream regions
SEQ ID NO:3—Mutant DNA sequence comprising upstream and downstream regions in contiguous configuration
SEQ ID NO:4—DNA sequence of middle region that is deleted in TH genes
SEQ ID NO:5—oligonucleotide sequence useful as a forward primer for PCR
SEQ ID NO:6—oligonucleotide sequence useful as a reverse primer for PCR
SEQ ID NO:7—oligonucleotide sequence useful as a third primer (reverse) for PCR
References
U.S. Pat. Nos. 6,759,192, 5,498,521, 6,013,444, 4,683,202, 6,225,093, 6,306,591
U.S. Pub. Nos. 2006/0063191; 20030203372
WO0246465, GB012566; GB0103156; GB0030076; AU0220920
Everts-Van der Wind et al., (2004)
Lapointe, J.-M., Lachance, S., and Steffen D. J. (2000). “Tibial Hemimelia, Menengocele, and Abdominal Hernia in Shorthorn Cattle.” Vet Pathol 37:508-511.
Leipold, H. W., Saperstein, G., Swanson, R., Guffy, M. M., and Shalles, R. (1978). “Inheritance of Tibial Hemimelia in Galloway Cattle.” Z Tierzuchtung Zuchtungbil 94:291-295.
Marron, B. M. et al. (January 2005) Mapping of the locus causing tibial hemimelia (TH) in shorthorn cattle. Plant & Animal Genomes XIII Conference. San Diego, Calif.
Ojo, S. A., Guffy, M. M., Saperstein, G., Leipold, H. W. (1974). “Tibial Hemimelia in Galloway Calves.” J Am Vet Med Assoc 165:548-550.







TABLE 1





Linkage analysis results generated by CRI-MAP. The number informative meioses for each marker is provided 


on the diagonal (italics). Two-point LOD scores are provided above the diagonal (bold), with those above 3 


highlighted in shading. 


The recombination frequencies between pairs of loci are provided below the diagonal (plain text).



























TABLE 2





Recombination events between th (dark) and normal haplotypes (white) of affected calves. 


The frequency of each haplotype is provided on the vertical axis, and ordered markers are 


provided along the horizontal axis. This graph suggests that the th locus lies between markers 6 and 7.



























TABLE 3A





SEQ ID NOs: 1-2, BOVINE EXT2 AND ALX4 GENES









































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































































TABLE 3B







Sequence no. identifiers for oligonucleotide sequences of Table 3









Seq. ID
Name in TABLE 3A
Sequence





Seq ID NO: 8
TH_MICRO243
ATATTGGCCAGGTCTTGATG


 


Seq ID NO: 9
TH_MICRO348C
AAGCTACAGACTGAGGGTTG


 


Seq ID NO: 10
EXT2_840C
AGTAGAACGAGCTTTGCCAC


 


Seq ID NO: ll
EXT2_744
ACTGGGAATTGCTGGCATAG


 


Seq ID NO: 12
TH_MICRO470C
ATGGACAGTGGAGCCTGAAA


 


Seq ID NO: 13
TH_MICRO332
TTCAGATTACTGCCAAGCCC


 


Seq ID NO: 14
TH_MICRO772C
TGATCCTGGTGATGGTTACA


 


Seq ID NO: l5
TH_MICRO573
CTTTGTCATGGACCAGCATC


 


Seq ID NO: 16
BALX4_4R
GGGAATCTAGCCTCAACTCA


 


Seq ID NO: 17
ALX4_EXON4
TGTGGCCCAGGAGATGGCCGCGCTGTGCTCCTTGGCCTTCATGCG




GAGGGCCGCGATGCTCGAAGTCTTGCGGTCCGGCTCCCCGTTGAG




CTCATAGCCGTTGAGGCCCGGGCTGAGGCCCGCAGCTCCAAACAG




GCTGCCCATGTGCGTCTGGCCCACGTGGCTGCCAGCCCCCGAGAC




GCTCAGGAAGTCGGTGACGCCGCTGGCCCCGGAGCCGGGGGGGTG




GGCGTGAGGGGACATGCAGGCGGGCACCGGGTCACAGGGCACCAC




ACAGGCCGGCACGGGCGAGGCGGCCCCATTGTTGCCGATCCAGGA




CGGGTTCTGAAT


 


Seq ID NO: 18
BALX4_4F
TTCCTGAGTGTTTACCTGGG


 


Seq ID NO: 19
BALX4_3R
CCTGGCCTTCAGCATTCCTC


 


Seq ID NO: 20
ALX4_EXON3
CTGGGCATAGTTTTCCGCTCGGGTGAGGAGGGGCAGCTCGTAGGC




TGTGGAGAAGTGGGTCCGAACCTGCTGCATTTGCCCGAAGCGTTC




CCTCTTCCTCCACTTGGCCCTCCGGTTCTGGAACCAGAC


 


Seq ID NO: 21
BALX4_3F
AGAGCAGTTGTCGCCAGGGT


 


Seq ID NO: 22
BALX4_2_1241C
GCAGGATGGGAACGTGAACA


 


Seq ID NO: 23
BALX4_2_889
TTGCTCTCCGAGTCGGCCTT


 


Seq ID NO: 24
BALX4_2_836C
CCAGCTTCCTTCACACTGAG


 


Seq ID NO: 25
ALX4_EXON2
CTGCACGCGGGCCTCGGTGAGGTCGGTCCTCATGGCCAGCTGCTC




CCGCGCGTACACATCGGGGTAGTGGGTCTTCTGGAAGACCTTCTC




CAGCTCCTCCAACTGGTAGCTGGTGAAGGTGGTTCTGTTCCGCCG




CTTCTTGCCCTTGTTGCTCTCCGAGTCGGCCTTCTCCATTGGGCT




GGGGAGGTCGGCGCTGGCCCGGTCCTGGGGCCCCTTCACCCCAGC




TTCCTTCACACTGAGGTAGCTGCTGTCCATCCCCACCGTGTCAGA




GTCGGGTGGCAACTCTGGCTCACCCAGGGAGCTCTCTTTGG


 


Seq ID NO: 26
BALX4_2_627
GAAGCATGGGATGCAAGGTC


 


Seq ID NO: 27
ALX4_2F
TGGGTGGGTCTGGACATGAGT


 


Seq ID NO: 28
ALX4_2R
CGTATGGAGCGTCTGTCTAAGG


 


Seq ID NO: 29
216232_R
GCTAGAATCCTGAATGGTGC


 


Seq ID NO: 30
216232_F
GAGACAGTGTCTAAGACGCA


 


Seq ID NO: 31
NW_216232_R2
GCTCTGTGCATACGATGAAG


 


Seq ID NO: 32
NW_216232_F2
ACATTCCACAGACCTCCGTC


 


Seq ID NO: 33
DEL5_699
GGCATGAAAGGAGGACTCTA


 


Seq ID NO: 34
DEL5_1109C
CGAGTCTGTTACCACTTCCT


 


Seq ID NO: 35
DEL5_1389
AAAGCATTGCCAAGTGTGGA


 


Seq ID NO: 36
DEL5_1754C
ATCAATTGTGAGGTTGCCAG


 


Seq ID NO: 37
DEL5_2006
AGCACAGTGCTTGTCAGGAA


 


Seq ID NO: 38
DEL5_2497C
CTGTCTGTCAAAGTTTCCGG


 


Seq ID NO: 39
DEL5_2843
CATTAGAAGCTGCGCCTGTT


 


Seq ID NO: 40
DEL5_3292C
ATCTTCAGGACATTCACGGG


 


Seq ID NO: 41
DEL5_3659
GGAGTTGAGCAGGATGACTT


 


Seq ID NO: 42
DEL5_5227C
AAGAGGCTGCACTAAGTCTG


 


Seq ID NO: 43
DEL5_5516
TAAGTCCCAGTCCATTCGCA


 


Seq ID NO: 44
DEL5_5967C
AGCGTCATCAGAAGAACCAG


 


Seq ID NO: 45
DEL5_6496
ATCAGAGGTTCTGGCACGAT


 


Seq ID NO: 46
DEL5_7040C
TATCAGGGAGGCCCACAAAC


 


Seq ID NO: 47
DEL5_7326
CAGGCAATTTCCAGGGAAGA


 


Seq ID NO: 48
DEL5_7683C
TGAGGAGCTGGAATTAGGCA


 


Seq ID NO: 49
DEL5_7991
GAGTTCCTGCAATCCCATCA


 


Seq ID NO: 50
TH_BIGBREAK_F
TGCTCAGGCTGGTTTCTCTTCC


 


Seq ID NO: 51
DEL5_8750C
AAGACTCTGGTGGTTGCACA


 


Seq ID NO: 52
TH_BIG_344C
ACGCTGCTGACTATACTGGGTG


 


Seq ID NO: 53
TH_BIG_478C
AGATGAGTGTGAGCCGGGAACA


 


Seq ID NO: 54
DEL5_9059
TCATAGCTTCCTTCCGGACA


 


Seq ID NO: 55
DEL5_9528C
ATGAAATGCGCACTGACTCC


 


Seq ID NO: 56
DEL5_9917
TACGGCGGCAGAATGTTTCA


 


Seq ID NO: 57
DEL5_10399C
ACTCTAAATTCGCTGGGTGG


 


Seq ID NO: 58
DEL5_10637
AGGCATTCTCCCAGATCCAT


 


Seq ID NO: 59
DEL5_10942C
ACAAAGCCGCACGCAGATTT


 


Seq ID NO: 60
CTG45_R2
GGTCTGTATCTCCATCCACG


 


Srq ID NO: 61
CTG45_F2
GGTTCTTAAGGCTAATGCGC


 


Seq ID NO: 62
CTG45_R1
CACAGAGGCCGACGGAAGAA


 


Srq ID NO: 63
CTG45_F1
TCAGAGGTGGTGCTCAGAGG


 


Seq ID NO: 64
BALX4_1_1162C
TTTAACAGCCAACGCTCCGTGC


 


Seq ID NO: 65
BALX4_1_1068C
CGACCCAAAGTCACAAACCGCT


 


Seq ID NO: 66
BALX4_1_1116C
ACCTGCAAGGCCGCGCTTGT


 


Seq ID NO: 67
BALX4_1_1061
CTGGAGTTTGAGGCTGCCGT


 


Seq ID NO: 68
BALX4_1_1042C
CGCCTCGTTGCAAGTAGAGA


 


Seq ID NO: 69
BALX4_1_851C/
GCTGACAGGAAAGTTGTGCTGA



BALX4_1_830



 


Seq ID NO: 70
ALX4_EXON1
CGTAGCAGGGGACCTGCAAGGCCGCGTTGTGGCCGCCGCCGCCT




TCCTGGAGTTTGAGGCTGCCGTCCGGGGGCGTCTTGCAGGCGCC




TCGTTGCAAGTAGAGATGCGGCTGCGGCGCGGGCGGCTGCGGCT




GCGGCGCGGGCGGCTGGGGCTGGAACTTGCTGAAGGAGCCCCGC




GCCCCGGCGCCGCTCTCCAGAGGTGCTGCCGGGTCCTGCTGCCC




CGCGCCGTAGCGGGCCCGGCTCTTGGCGTCCCCGAATCCCTGCC




CTTTGGCGGCGGCTGACAGGAAAGTTGTGCTGAACTTATCACCG




CCGGGGTATGCCCTAAAAGGCGACGAGCCCTCCCGGCTCTGCGA




CACCGGGCTGTAGTAGGCGTCCATGGCAGCAGCCGGCGACTCGC




AGTAAGAGACGCAAGTCTCAGCATT


 


Seq ID NO: 71
BALX4_1_428
AGTTGACCAGATCTCCCAAACCCT


 


Seq ID NO: 72
BALX4_1_426
AGTTGACCAGATCTCCCAAACCCTCTT


 


Seq ID NO: 73
BALX4_1_45
ATTCGGTCGCCACTCC


 


Seq ID NO: 74
CTG60_Fl
GGAGTTGAGAGACCCGCACC


 


Seq ID NO: 75
ALX4_68_249
GGAACAGGACTTCACTACAG


 


Seq ID NO: 76
CTG60_Rl
CCTTCTACACGGCTAATCAC


 


Seq ID NO: 77
ALX4_68_1421C
CATCGCACTAGTCTGAGGTT


 


Seq ID NO: 78
320933_F2
GGAATGCAGACACCTAGTCA


 


Seq ID NO: 79
320933_R2
GTTCTAGACGCTTGCACAAC


 


Seq ID NO: 80
ALX4_33_144
AGCTCTAGGAAGTTGACAGG


 


Seq ID NO: 81
ALX4_33_708C
GGTCCAGACTGTCACAGATG


 


Seq ID NO: 82
370933_Rl
CCTAAGGAGATGAGAAGCAG


 


Seq ID NO: 83
320933_Fl
GTCCTGATCTCTGATGTGCC


 


Seq ID NO: 84
NW_419640_Rl
TTGGCTCCGCTGTCTAGAAT


 


Seq ID NO: 85
NW_419640_Fl
TCATCTTGCTGCATGTCGGT


 


Seq ID NO: 86
NW_250796_R2
CAGCTTCCTGAGTGTGATCC


 


Seq ID NO: 87
NW_250796_F2
CATACAGACCTGGCTCAAGT


 


Seq ID NO: 88
NW_250796_Rl
ACGCTCTGGATCTCATCCTT


 


Seq ID NO: 89
DEL3_308
CTTAGCTCCAGAGATGCGAA


 


Seq ID NO: 90
NW_250796_Fl
AGTCACCTGCTACCATCTCC


 


Seq ID NO: 91
TH_BIG_895/
CCGAACTGAACTGACACTTGCT



DEL3_2389



 


Seq ID NO: 92
DEL3_2532C
ACCTCTATGCCACCTAGGAT


 


Seq ID NO: 93
DPL3_2671
AAGATGAGTCCTTGGCCACA


 


Seq ID NO: 94
TH_BIGBREAK_R
ACGAGAGGCCTTGTCTTTGCAC


 


Seq ID NO: 95
DEL3_2952C
TCAGTGCCTTCCTCCTCATA


 


Seq ID NO: 96
DEL3_3269
TGATCCTGGACTCAGTGATG


 


Seq ID NO: 97
DEL3_3681C
ACAGATTGGAACGAGGGAGA


 


Seq ID NO: 98
DEL3_4068
AGTAAGCCCTCTTGAAGCCA


 


Seq ID NO: 99
DEL3_4428C
TAGAGCTTGAGGTGCCTTGA


 


Seq ID NO: 100
NW_197235_Rl
AACACAGCACACAGATCGGA


 


Seq ID NO: 101
NW_197235_Fl
CTGCATGCTGATTAGCTGGT


 


Seq ID NO: 102
288353_R2
TTCTGAACTGGCTCTTGTGG


 


Seq ID NO: 103
288353_F2
TTCAGCCTGTACCTCAGTGA
















TABLE 4





SEQ ID NO: 3, 


TIBIAL HEMIMELIA DELETION BREAKPOINT REGION















GTTTAACGAATTCGCCCTTCAGGCAATTTCCAGGGAAGAGCCAACACATG


 


AACCCAGAGCTCTTGATGTCCAGAGAAGCCATGGAGTGGGGAAGGAGAAA


 


GCAAAGAAGCAGCTACTCAGCCACCAGAGAAGATCCAGGTGGTCTGTGGC


 


AACTCTAAGCCCTCCCCTTACGGGGTAAAGCTCCACCCACCTGGGAGCAG


 


GCTGCCCTTCTCCGCCCAGCCTCTCCAGCACCAATCCATTAACTGCAGTG


 


AACCAAGCTACTAGTCTGTCTCCCCATCCCGACCTGAAGGCAGAGGCTGT


 


GTCCAGGGCCTCACAGAATCCCCCAGGAGGCCAGGAACAGGGACAGGCAA


 


AGTCTGGTGAGGAGCTGGAATTAGGCATTTCAGTCCCCTTCTCTGTGAAA


 


ACTGGGCATTTGGGCCAGGAGGCGTCTGGCGTCTCTTCCAACACTGGGCG


 


GGACACCCATCCCGACACCAGGACCCATCATGTTGGAGGGTTGACTTCCG


 


GGCCTCAACCAAGGTCCCCGACCTGGCAGGTGGTCAGCCTCAGAGGAGGA


 


AATAAGTCATGTGGCCTCAGCTAACACCCCTTGGGCTCTCACTGCCGGGT


 


TTCCACACACTTAGGAAACAAAACCCCGGGTCAGGGAGCCCAGCAGAGAG


 


CAGGAGTCCCTGCCCCACTGGGTCACATTTGGGGATGAGTTCCTGCAATC


 


CCATCAGGTGCTCTTCTGTTGCCCTGGCAACCCCAGGAGCTCCCAGTGGA


 


GCCCATCTCATTTCTGAAGGAGGGGGAAAGGGCAAGCATCTGCTGTGCAT


 


GAGACACAGAGTGTGTGGCCAACTGGATGGATTACAGTGAAAGGAGACGG


 


TAGGAGAAGGGGCACAGGTACCCCAGCTCCTTCTTCCTTCTCCACCCTCT


 


GCAGTCCTTCCTTCCTTCCCACTGACTTGCCTGTGGGGTTCCATTCCCCT


 


TTAGGCCTCAGTTTGTTGTTGATTAGTGGCTAGGTCGTGTCCGACTCTTG


 


CAACCCTGTGGACTGTAGCCTGCCTCTGTCCATGGGATTTTCCCAGGCAA


 


GAATACTGGAGGGGGTTGCCATTTCCTTCTCCATGGGGGTCTTCCTGACC


 


CAGGGATCGAGCCCCAGCCCGCGTCTCCTGCATCAGCGGGTGGGTTCTTT


 


ACCACTGAGCCACCAGAGAAGCCCAGGCCTCCGTTTGCCAAGACGGTGCT


 


CAGGCTGGTTTCTCTTCCTCAGAACTGAGGAAATGCTCAAGAGCTGGAGG


 


TGGGAGGAGGCCCCACTGCG GTCCTCAAGGGCAGGACCTCTATGCCACC


 


TAGGATGAAGGCTCCCCACGCCCACACCTCCCATCCCTTTGATGCCTGGA


 


GGGACAGGAAGCAGGGTGGCAAGATGGTGCCTCTGGTCTAGTCCACACCC


 


CACACCCCTGGTTTGGTGGTGAGGTGGCCCCATGCCTGACCAGGAAGATG


 


ACTCCTTGGCCACAGACTGGCCTCGCTGCATCCTCTGCTCCCTACCTCTC


 


CTCCTGAGGTCCCTGGGGGTGGGGGGAATTGGTGGCCTCCTAGGAAAGAA


 


CCCACTTCATCTGTACTACAGATACCACCCCCGACCCTGAGACCACGAGA


 


GGCCTTGTCTTTGCACCTTAACACACCTCGTCTTTGCTCCTCTGTCTGCC


 


CTGCCCCCAGCCTCCATTGGTTTGACTCCTCAGTGCC





 Indicates position of deleted DNA segment of 45,694 base pairs













TABLE 5





BLAST ANALYSIS OF SEQID 2 WITH SEQID 1





















 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 






















TABLE 6







Summary of PCR product generated by


three primers to determine TH status











Normal (−/−)
Hetero Carrier (+/−)
TH phenotype





Primer 1-2 (L12)
−
+ (from mutant allele)
+


Primer 1-3 (L13)
+
+ (from normal allele)
−





− no amplified DNA product generated by PCR


+ amplified DNA product generated by PCR


Primer 1: primer binds upstream region


Primer 2: primer binds downstream region


Primer 3: primer binds middle (e.g. mutant) region

## Claims

1. A method of diagnosing a deletion mutation in a bovine genome, said method comprising:
a) obtaining a DNA sample from the bovine; and
b) analyzing said DNA sample to determine the presence or absence of a deletion mutation in the Aristaless-like4 (ALX4) gene, wherein said deletion mutation comprises deletion of SEQ ID NO:4 and said deletion mutation is at a breakpoint between nucleotides 1270 and 1271 of SEQ ID NO:3.

2. The method of claim 1, wherein said deletion mutation results in a mutant bovine gene comprising SEQ ID NO:3.

3. The method of claim 1, further comprising identifying the
DNA sample as from a heterozygous carrier of the Tibial hemimelia (TH) gene.

4. The method of claim 1, wherein the step of analyzing the DNA sample is by polymerase chain reaction (PCR).

5. The method of claim 4, wherein the step of PCR further comprises:
a) providing a forward primer which binds specifically to a first selected region of SEQ ID NO:2, said first region between base 27,000 and 29,692 of SEQ ID NO:2;
b) providing a reverse primer which binds specifically to a second selected region of SEQ ID NO:2, said second region between base 75,386 and 78,000; and
c) performing PCR amplification such that said forward and reverse primer generate an amplified DNA product only in the presence of a DNA sample comprising the deletion mutation.

6. The method of claim 5, wherein the forward primer has a sequence that is SEQ ID NO:5 and the reverse primer has a sequence that is SEQ ID NO:6.

7. The method of claim 5 further comprising providing a third primer, said third primer capable of specific binding to at least a portion of SEQ ID NO:3 and corresponding to a DNA sequence encompassed by bases 29,693 to 75,385 of SEQ ID NO:2, wherein sample DNA that contains SEQ ID NO:3 results in amplification of a first DNA product by said forward primer and said third primer, and wherein DNA that does not contain said SEQ ID NO:3 results in amplification of a second DNA product by said forward primer and said reverse primer, and wherein said forward primer and said third primer binding position are separated by a separation distance that is less than or equal to 4000 base pairs.

8. The method of claim 7, wherein the third primer has a sequence that is SEQ ID NO:7.

9. The method of claim 8, wherein the forward primer has a sequence that is SEQ ID NO:5, and the reverse primer has a sequence that is SEQ ID NO:6.

10. The method of claim 4 further comprising providing a forward primer and a reverse primer, wherein said forward primer and said reverse primer are capable of specific binding to said DNA sample to generate an amplification DNA product only if the DNA sample comprises SEQ ID NO:4.

11. The method of claim 1 further comprising
a) providing a probe or primer that specifically binds to the sample of DNA having said deletion mutation; and
b) identifying the sample as containing the deletion mutation for DNA samples that hybridize with said probe or primer;
wherein said specific binding is at a location that includes nucleotides 1270 and 1271 of SEQ ID NO:4.

12. The method of claim 1, wherein the analyzing the DNA sample further comprises providing a DNA probe or primer, wherein said DNA probe or primer specifically binds to SEQ ID NO:4 and does not specifically bind to bovine DNA comprising SEQ ID NO:3.

13. The method of claim 1, wherein the analyzing the DNA sample further comprises providing a DNA probe or primer, wherein said DNA probe or primer specifically binds to bovine DNA comprising SEQ ID NO:3 and does not specifically bind to bovine DNA comprising SEQ ID NO:4.

14. The method of claim 1, wherein said analyzing the DNA sample comprises DNA sequencing.

15. The method of claim 1, wherein said analyzing the DNA sample comprises providing a probe or primer comprising a purified oligonucleotide, wherein the oligonucleotide has a length between about 15 to 50 nucleotides and specifically binds to a target sequence of SEQ ID NO:4 or complement thereof.

16. The method of claim 1, wherein said analyzing the DNA of the bovine subject comprises providing a probe or primer comprising a purified oligonucleotide, wherein the oligonucleotide has a length between about 15 to 50 nucleotides and specifically binds a breakpoint mutation region in the bovine genome, said breakpoint located between bases 1270 and 1271 of SEQ ID NO:3.

17. The method of claim 1, wherein the bovine is a Shorthorn composite.

18. The method of claim 17, wherein the DNA sample is obtained from blood or semen.

19. A method for screening a bovine to determine if said bovine carries the gene for TH, said method comprising:
a) providing a biological sample which was removed from said bovine to be screened; and
b) conducting a biological assay to determine the presence of a mutation in the gene responsible for TH, wherein the mutation comprises a deletion of bases 29,693 to 75,385 in SEQ ID NO:2.

20. A method of diagnosing a mutation in a bovine genome, wherein said mutation is associated with tibial hemimelia (TH), and wherein the mutation is a deletion mutation of SEQ ID NO:4 in the gene that encodes Aristaless-like4 (ALX4) protein, said method comprising:
obtaining a DNA sample from the bovine; and
analyzing said DNA sample to determine the presence or absence of the deletion mutation of SEQ ID NO:4 at a breakpoint between nucleotides 1270 and 1271 of SEQ ID NO:3 in the ALX4 gene, wherein said DNA having an upstream region, a downstream region, and a middle region that comprises SEQ ID NO:4 that separates said upstream region from said downstream region indicates absence of the deletion mutation, and wherein said DNA having said upstream region and said downstream region in a contiguous configuration by deletion of SEQ ID NO:4 is identified as having the deletion mutation;

wherein the step of analyzing the DNA sample is by polymerase chain reaction further comprising the steps of:
providing a forward primer which binds specifically to a first selected region of SEQ ID NO:2, said first region between base 27,000 and 29,692 of SEQ ID NO:2;
providing a reverse primer which binds specifically to a second selected region of SEQ ID NO:2, said second region between base 75,386 and 78,000;
performing PCR amplification such that said forward and reverse primer generate an amplified DNA product only in the presence of a DNA sample comprising the deletion of SEQ ID NO:4 from the bovine genome; and
providing a third primer, said third primer capable of specific binding to at least a portion of the middle region of DNA, said middle region corresponding to a DNA sequence encompassed by bases 29,693 to 75,385 of SEQ ID NO:2, wherein DNA that contains said middle region results in amplification of a first DNA product by said forward primer and said third primer, and wherein DNA that does not contain said middle region results in amplification of a second DNA product by said forward primer and said reverse primer, and wherein said forward primer and said third primer binding positions are separated by a separation distance that is less than or equal to 4000 base pairs.

21. The method of claim 20, wherein the forward primer has a sequence that is SEQ ID NO:5, the reverse primer has a sequence that is SEQ ID NO:6, and the third primer has a sequence that is SEQ ID NO:7.

22. The method of claim 20, wherein the forward primer has a sequence that is SEQ ID NO:5, and the reverse primer has a sequence that is SEQ ID NO:6.

23. The method of claim 7, wherein:
said forward primer is selected from the group consisting of: TH_BIGBREAK_F (SEQ ID NO:5), DEL5—7991 (SEQ ID NO:49; nucleotides 29109 to 29128 of SEQ ID NO:2), DEL5—7683C (SEQ ID NO:48; nucleotides 28782 to 28801 of SEQ ID NO:2), DEL5—7326 (SEQ ID NO:47; nucleotides 28444 to 28463 of SEQ ID NO:2), DEL5—7040C (SEQ ID NO:46; nucleotides 28139 to 28158 of SEQ ID NO:2), DEL5—6496 (SEQ ID NO:45; nucleotides 27614 to 27633 of SEQ ID NO:2) and DEL513 5967C (SEQ ID NO:44; nucleotides 27091 to 27110 of SEQ ID NO:2);
said reverse primer is selected from the group consisting of the reverse complement to the sequence of: DEL3—2532C (SEQ ID NO:92; nucleotides 75401 to 75420 of SEQ ID NO:2), DEL3—2671 (SEQ ID NO:93; nucleotides 75559 to 75568 of SEQ ID NO:2), TH_BIGBREAK_R (SEQ ID NO:94; nucleotides 75709 to 75730of SEQ ID NO:2), DEL3—2952C (SEQ ID NO:95; nucleotides 75794 to 75813 of SEQ ID NO:2), DEL3—3269 (SEQ ID NO:96; nucleotides 76157 to 76176 of SEQ ID NO:2), DEL3—3681C (SEQ ID NO:97; nucleotides 76550 to 76569 of SEQ ID NO:2), DEL3—4068 (SEQ ID NO:98; nucleotides 76956 to 76975 of SEQ ID NO:2), DEL3—4428C (SEQ ID NO:99; nucleotides 77297 to 77316 of SEQ ID NO:2), NW—197235_R1 (SEQ ID NO:100; nucleotides 77626 to 77645 of SEQ ID NO:2) and NW—197235_F1 (SEQ ID NO:101; nucleotides 77895 to 77914 of SEQ ID NO:2); and
said third primer is selected from the group consisting of the reverse complement to the sequence of: DEL5—8750C (SEQ ID NO:51; nucleotides 29849 to 29868 of SEQ ID NO:2), TH_BIG—344C (SEQ ID NO:52; nucleotides 29941 to 29962 of SEQ ID NO:2), TH_BIG—478C (SEQ ID NO:53; nucleotides 30075 to 30096 of SEQ ID NO:2), DEL5—9059 (SEQ ID NO:54; nucleotides 30177 to 30196 of SEQ ID NO:2), DEL5—9528C (SEQ ID NO:55; nucleotides 30627 to 30646 of SEQ ID NO:2), DEL5—9917 (SEQ ID NO:56; nucleotides 31035 to 31054 of SEQ ID NO:2), DEL5—10399C, (SEQ ID NO:57; nucleotides 31498 to 31517 of SEQ ID NO:2) DEL5—10637 (SEQ ID NO:58; nucleotides 31755 to 31774 of SEQ ID NO:2) and DEL5—10942C (SEQ ID NO:59; nucleotides 32041 to 32060 of SEQ ID NO:2).

24. The method of claim 7, wherein said first amplified DNA product and said second amplified DNA product have different nucleotide lengths.