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Class i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencingRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Nucleic AcidClass i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencing description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070128629, Class i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencing. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. Ser. No. 10/632,571, filed Jul. 28, 2003, which is a continuation of U.S. Ser. No. 09/846,826, filed May 1, 2001; which is a continuation of U.S. Ser. No. 09/021,892, filed Feb. 11, 1998, now U.S. Pat. No. 6,287,764, issued Sep. 11, 2001; which claims the benefit of U.S. Provisional application Ser. No. 60/037,054, filed Feb. 11, 1997. BACKGROUND OF THE INVENTION [0003] 1. Field of the Invention [0004] The present invention relates in general to a method for direct DNA sequencing of HLA-A, -B, and -C alleles and more particularly the invention entails the sequence based typing of exons 2 and 3 for the HLA allele gene under study. [0005] 2. Brief Description of the Related Art [0006] MHC has the highest genetic polymorphism of the mammalian DNA molecules. Questions are raised by this polymorphism, such as its molecular basis, degree, and functional significance. For analysis of MHC polymorphism and its relationship to immune responses and disease susceptibilities, the human species has considerable scientific advantages, as well as direct relevance to clinical medicine. Only in human populations is there likely to be extensive analysis of MHC polymorphism from many geographically separated populations. The crystal structure of the human class I molecule has also been previously disclosed, making accurate insights into other HLA class I molecules possible as well as the allelic polymorphism of the HLA-A, B and C genes. [0007] The HLA class I genes are a component of the human major histocompatibility complex (MHC). The class I genes consist of the three classical genes encoding the major transplantation antigens HLA-A, HLA-B and HLA-C and seven non-classical class I genes, HLA-E, HLA-F, HLA-G, HLA-H, HLA-J, HLA-K, and HLA-L. [0008] The classical HLA class I genes encode polymorphic cell surface proteins expressed on most nucleated cells. The natural function of these proteins is to bind and present diverse sets of peptide fragments from intracellularly processed antigens to the T cell antigen receptors (TCRs). Thus, the peptide-binding capacity of the MHC molecule facilitates immune recognition of intracellular pathogens and altered self proteins. Therefore, by increasing the peptide repertoire for TCRs, the polymorphism of MHC molecules plays a critical role in the immune response potential of a host. On the other hand, MHC polymorphism exerts an immunological burden on the host transplanted with allogeneic tissues. As a result, mismatches in HLA class I molecules are one of the main causes of allograft rejection and graft versus host disease, and the level of HLA matching between tissue donor and recipient is a major factor in the success of allogeneic tissue and marrow transplants. It is therefore a matter of considerable medical significance to be able to determine the "type" of the HLA class I genes of candidate organ donors and recipients. [0009] HLA class I histocompatibility antigens for patient-donor matching are conventionally determined by serological typing. Biochemical and molecular techniques have revealed that HLA class I polymorphism is far greater than previously recognized by conventional methods. To date, over 59 HLA-A, 127 HLA-B, and 36 HLA-C different allelic sequences have been identified. This high level of allelic diversity complicates the typing of the HLA class I genes. [0010] Another complicating factor is the large number of homologous genes and alleles. Each of the HLA class I genes is composed of eight exons and seven introns and the sequences of these exons and introns are highly conserved across the HLA class I genes. Allelic variations mostly occur in exons 2 and 3 which are flanked by noncoding introns 1, 2, and 3. These two exons encode the functional domains of the molecules. [0011] Taken together, these two complications make HLA class I typing at the nucleic acid level a formidable task. Allelic diversity within any one gene means that a great many probes need to be developed if hybridization-based tests are used in the typing. Further, the general applicability of DNA typing methods to HLA class I genes depends on the design of primers which provide effective locus-specific amplification of exons 2 and/or 3 of one HLA class I gene. [0012] One method for performing HLA class I typing is disclosed in U.S. Pat. No. 5,424,184 which is incorporated herein by reference. This patent utilizes primers which are located within exons 2 and 3 of the HLA class I genes to achieve what is described as group-specific amplification of a portion of the HLA-A, HLA-B, and HLA-C genes. This approach is not ideal, however, since the primers hybridize with portions of the coding strand, and thus may mask significant allelic variations. In addition, this method requires a grouping of alleles by means of another method in order to select group-specific primers for amplification. [0013] Prior to inclusion of polymerase chain reaction (PCR) into the cloning and sequencing of HLA class I alleles the accumulation of class I sequences was a cumbersome act. The advent of the PCR greatly accelerated the accumulation of class I HLA sequences. Since then, research has been undertaken to bring molecularly-based HLA class I and class II typing techniques to the point where they would be clinically useful and cost-beneficial for the many applications such as bone marrow donor registry and disease association studies. [0014] Using the bone marrow donor registry as an example, the impetuses driving the development of new semi-automated and automated molecular techniques for high-resolution class I and class II typing could be characterized as: (1) the pool of available donors in the registry were poorly HLA typed: many HLA class I types are of low resolution and many clinically deleterious class I types were missed; (2) questions pertaining to discriminatory power of serology had to be addressed: serological HLA typing does not correlate with a high degree of accuracy with the molecular typing of HLA alleles; (3) it is recognized that continual HLA typing will be required to establish and maintain a donor registry of sufficient size; and (4) typing methods based on DNA sequence have many advantages over conventional serologic typing techniques including the elimination of typing complications due to different tissue distributions of MHC antigens, the use of defined reagents available in unlimited quantities (in contrast to alloantisera which are chemically ill-defined, limited in amount, and frequently monopolized), and the ability to provide an absolute HLA type. [0015] For HLA class II molecules, high resolution clinical typing using the polymerase chain reaction (PCR) with sequence specific primers (SSP) and/or sequence specific oligonucleotide probes (SSOP) is now a routine procedure. This advance in class II typing was essential because alloantisera, traditionally used for class I typing, proved especially inadequate for typing class II antigens. An additional advantage of DNA based HLA typing is that viable cells are not required. [0016] An increasing appreciation of the complexity and the extent of HLA class I polymorphism has consequently developed. The shortcomings of current class I serological typing procedures became more apparent, as did the need for a high resolution molecular typing technique. As a logical extension of methods successfully employed for class II typing several groups have applied PCR/SSP and/or PCR/SSOP methodologies to molecularly type HLA class I alleles. At first these applications were most successful for the subtyping of class I antigens for which there are multiple variants (e.g. A2, B27). These techniques subsequently began to prosper for the typing of all alleles at the HLA-A, and C loci, with the HLA-B locus providing the most resistance to these typing methodologies. [0017] The nucleic acid sequencing of class I HLA molecules has revealed numerous serological typing inadequacies, and the availability of cell lines containing sequenced class I alleles has been instrumental for the development of PCR primer and probe based typing techniques. Well characterized cell lines serve as important developmental standards and controls for these arriving class I molecular typing techniques. It is anticipated that this will hold true for emerging HLA-B typing protocols. [0018] Attempts to eliminate cloning from the determination of HLA class I sequences has been undertaken. To obviate cloning for direct sequence based class I typing three difficulties arise. The first difficulty is that sequence based typing must frequently resolve two nucleotides at one rung of the sequencing ladder; heterozygosity is the norm, and two alleles at one locus may differ by as many as 85 nucleotides. We therefore sought a DNA sequencer designed to utilize a single fluorophore, eliminating complications which might arise from different fluorophores simultaneously fluorescing in the same lane at heterozygous positions. The second difficulty is the occurrence of band compressions in the sequencing ladder due to the high G/C content of class I molecules. Past experience dictates that optimal resolution of G/C band compressions is obtained with a T7 sequencing chemistry utilizing 7-deaza dGTP. A third difficulty encountered by all who type class I HLA molecules is their polymorphic nature; once the class I DNA sequence is obtained, it is often difficult to assign a class I type to the data generated. [0019] The problem addressed is that for the successful transplantation of organs and bone marrow, for the proper diagnosis of autoimmune disorders such as arthritis, and for research studies trying to establish a link between a particular disease and immune response genes, an accurate class I HLA type must be established. However, clinical HLA class I typing laboratories (which now use antibodies for typing) cannot accurately discriminate among the many different class I genes found in the population. Therefore, molecular DNA based methods are being tested to facilitate more precise HLA class I typing. [0020] Others are testing molecular DNA class I HLA typing methodologies. Some rely on the failure or ability to PCR amplify a gene, with several hundred PCR reactions needed to call a class I type. This is termed SSP (sequence specific PCR). Other techniques utilize one HLA-A, -B, and -C locus specific PCR reaction followed by hybridization with a complex series of oligonucleotide probes. This technique is referred to as SSOP. These techniques utilize a similar first step--an HLA-A, -B, and -C locus specific PCR reaction--followed by divergent methods for typing the HLA-A, -B, or -C specific PCR product. [0021] A comparison of SSP, SSOP, and the present invention which we will term "sequence based typing" (SBT) shows that SBT gives a precise class I HLA type, while SSP and SSOP give only a partial type, i.e. they probe portions of the genes being typed while SBT reads all of the gene, being typed. The reason others have been reluctant to adopt a sequence based typing approach is because the technology is complex and developing. [0022] Due to the fact that SBT gives the most precise class I type, several other groups have tried to develop a class I HLA SBT method. What individuates the techniques described herein is: (1) amplification of HLA -A, -B, or -C class I alleles such that exons 2 and 3 are produced in a locus specific way to facilitate (2) production of a secondary HLA -A, -B, or -C locus specific polymerase chain reaction by separately amplifying exons 2 and 3 using the primary polymerase chain reaction product as a template and nested or heminested polymerase chain reaction primers and (3) preparing the secondary polymerase chain reaction product such that it has an anchoring moiety at one terminus and a DNA sequencing primer site at the opposing terminus and (4) following attachment to a solid support the secondary polymerase chain reaction products are then DNA sequenced. [0023] Thus, the novel aspect of our approach is that HLA -A, -B, or -C locus specific nested polymerase chain reaction products are produced at a level of purity sufficient for DNA sequence based typing. Furthermore, these secondary polymerase chain reaction products can be anchored to a solid support prior to DNA sequencing with a universal DNA sequencing primer. Continue reading about Class i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencing... Full patent description for Class i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencing Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Class i sequence based typing of hla-a, -b, and -c alleles by direct dna sequencing patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. 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