US 5,629,149 AGrant
DNA Sequence-Based HLA Typing Method
Issue Date:1997-05-13
•36 Claims
•4 Drawing Sheets
Abstract
The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class II and Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of DRB, DQB, DQA, DPB, DPA, HLA-A, HLA-B and HLA-C- transcripts enzymatically amplified using a limited number of non-allele-specific oligonucleotides. Total cellular RNA from peripheral blood mononuclear cells is reverse transcribed using antisense primers, specific for different locus (DQB, DQA, DPA or DPB) or group of loci (DRB1-5, or HLA-A and HLA-B and HLA-C). The synthesized cDNA molecules are then enzymatically amplified using different combinations of oligonucleotides for each locus and directly sequenced with Taq polymerase using an internal oligonucleotide. The sequenced genes are then analyzed.
Metadata
Assignee
- Regents of the University of Minnesota
Inventors
- Pedro Santamaria
- Michael T. Boyce-Jacino
- Jose J. Barbosa
- Stephen S. Rich
- Anthony J. Faras
Application Information
Application Number:US 1068024
Filing Date:1993-08-16
Priority Date:1991-03-06
Art Unit:184
Classifications
IPC:
C12Q 168C12Q 148C12N 1511
Field of Search:
4355369356;15;91.1;91.2;91.5;91.5124.338;17;18;77;78
Patent Drawings (4 sheets)
Description
TECHNICAL FIELD
The present invention relates to a process for determining genotypes of highly polymorphic systems, such as the major histocompatibility complex of humans, including Class I and Class II HLA genes. Specifically, the method of the present invention involves amplifying the alleles carried by any given individual at a gene locus or loci of interest by polymerase chain reaction with conserved and non-conserved oligonucleotide primers. The polymerase chain reaction products are directly sequenced followed by evaluation of the resulting nucleic acid ladders to determine the genotype of sample nucleic acid.
BACKGROUND OF THE INVENTION
The major histocompatibility complex (MHC) includes the human leukocyte antigens (HLA) gene complex which is located on the short arm of human chromosome six. This region encodes cell-surface proteins which regulate the cell-cell interactions of the immune response. The various HLA Class I loci encode the HLA antigens, 44,000 dalton polypeptides which associate with B-2 microglobulin at the cell surface. The Class I molecules are involved in the recognition of target cells by cytotoxic T lymphocytes. HLA Class II loci encode cell surface heterodimers, composed of proteins of 29,000 and 34,000 daltons, respectively. These Class II molecules are also involved in the recognition of target cells by helper T lymphocytes.
The HLA-A, HLA-B, and HLA-C loci of the HLA Class I region as well as the HLA-DRB, HLA-DQB, HLA-DQA, HLA-DPB and HLA-DPA loci of the HLA Class II region exhibit an extremely high degree of polymorphism. The WHO nomenclature committee for factors of the HLA system [Marsh and Bodmer, Immunogenetics, 31:131 (1990)] designated 25 alleles of HLA-A (HLA-A-0101, A-0201, etc.), 32 alleles of HLA-B, and 11 alleles of HLA-C, 43 HLA-DRB alleles, 13 HLA-DQB alleles, 8 HLA-DQA alleles, 4 HLA-DPA alleles and 19 HLA-DPB alleles. Since this high degree of polymorphism is thought to relate to the function of the HLA molecules, much effort has gone into determining its molecular basis and the functional implications of its polymorphisms (i.e., in transplantation). With the cloning of certain HLA genes this effort has extended to the DNA level.
The Class II genes of the HLA-D region on the short arm of human chromosome six constitute one of the most polymorphic genetic systems known [Bach, Immunol. Today, 6:89 (1985)]. The HLA Class II molecules (DR, DQ and DP) are heterodimeric glycoproteins composed of two non-covalently associated chains (alpha and beta) which serve as restricting elements in nominal antigen presentation in the context of self [Zinkernagel and Doherty, Nature, 248:701 (1974)] or as foreign antigens in alloresponses [Bach and Van Rood, N. Engl. J. Med., 295:806 (1976)].
Allelic polymorphism of the HLA-D region encoded specificities can be determined by serological methods for phenotyping, mixed lymphocyte cultures using homozygous typing cells, primed lymphocyte testing, determination of restriction fragment length polymorphisms and, more recently, oligotyping [Bach, supra (1985); Bidwell, Immunol. Today, 9:18 (1988); Tiercy et al., Proc. Natl. Acad. Sci. USA, 85:198 (1988)]. Present efforts focus largely on the development of molecular approaches to typing, such as RFLP and oligotyping [Bidwell, supra (1988); Tiercy et al., supra (1988); Erlich and Bugawan, in PCR Techniques, H. A. Erlich, ed., Stockton Press, New York (1989)].
The cloning and sequencing of several HLA-DR, DQ, and -DP alleles has revealed that their amino acid polymorphisms are located in hypervariable regions of their N-terminal domains, encoded by the second exon of DRB1, DRB3/4/5, DQA1 and DQB1, DPA1 and DPB1 genes [Marsh and Bodmer, supra (1990); Todd et al., Nature, 329:599 (1987)]. This information has allowed the design of allele-specific oligonucleotides which can be used in the characterization of the known HLA Class II polymorphisms by means of their hybridization to DNA on a solid support (oligomer typing) or for sequencing [Tiercy et al., supra (1988); Erlich and Bugawan, supra, (1989); Todd et al., supra (1987); Saiki et al., Science, 230:1350 (1985); Mullis and Faloona, Methods Enzymol., 155:335 (1987); Saiki et al., Nature, 324:163 (1986); Scharf et al., Science, 233:1076 (1986); Gyllenstein and Erlich, Proc. Natl. Acad. Sci. USA, 85:7652 (1988)]. Oligonucleotide typing, although rapid, requires the use of a rather large number of oligonucleotides for each locus and cannot detect previously unidentified sequence polymorphisms, likely to exist in non-Caucasian populations; further, the approach may not be easily applicable to and may not be practical for the analysis of Class I polymorphisms. Direct sequencing of single-stranded DNA generated by PCR using allele-specific oligonucleotides has been successfully used to examine polymorphism at DQA1 locus [Gyllenstein and Erlich, supra (1988)]. Application of this approach to DRB genes is, however, problematic due to the strong sequence homology among DRB1, DRB3, DRB4 and DRB5 genes and the presence of up to four different versions of each of these genes in most individuals (isotypic complexity). The very complex ladders generated by direct sequencing make this present process impractical for accurate and rapid determination of HLA types. Thus, direct sequencing of HLA-PCR products has been limited to previous knowledge of the HLA types carried by a given individual and as such is not suitable for routine HLA typing [Bach, supra (1985); Bidwell, supra (1988); Tiercy et al., supra (1988); Erlich and Bugawan, supra (1989)].
Currently, HLA typing is routinely done in connection with many medical procedures, e.g., organ transplantation. Rejection of organ grafts is believed to be diminished if the HLA alleles of donor and recipient are identical. The numerous alleles of HLA genes in the population also make HLA typing useful for paternity testing. However, the currently available techniques are incapable of differentiating among all of the polymorphisms associated with the alleles at Class I and Class II HLA loci. Other drawbacks to current HLA typing are the availability of standard sera necessary to conduct serological tests, the speed of obtaining test results (i.e., MLC takes 5-7 days), and that only the already known HLA types, but not new polymorphisms, are detected by these techniques. In the case of tissue typing in organ transplants and in relatively high volume genetic evaluations, such as paternity testing, the length of time associated with current HLA typing techniques causes unnecessary delay and the results may not be highly accurate.
Accordingly, there is a need for a method to determine genomic information in highly polymorphic systems, such as the HLA gene complex, that addresses the limitations imposed by previous methods. That is, in the case of the HLA gene complex, a system that is capable of determining the nucleotide sequences of the genes carried by any given individual without the need to have previous knowledge of his or her HLA types as defined by other methods. Furthermore, the invention avoids the use of oligonucleotides specific for each known allele. The technique we present is rapid, requires the use of only a small number of oligonucleotide primers, and can readily detect new sequence variants unidentifiable with more conventional approaches. This system is exemplified by its applicability to the analysis of Class II as well as Class I and Class III genes and is automatable.
SUMMARY OF THE INVENTION
The present invention relates to a method for determining the nucleic acid sequence of one or more polymorphic genes of a subject by amplifying and direct sequencing genomic or complementary DNA molecules for each allele at each gene locus to be sequenced using conserved and non-conserved (non-allele-specific) oligonucleotide primers. In a broad sense, the method of the present invention involves sequence-based typing (SBT) which provides for unequivocal determination of genetic polymorphism at any genetic locus of interest by direct, simultaneous, sequence analysis of both genomic DNA or expressed (RNA) copies of such a locus. SBT can be employed to determine genetic polymorphism at one or more genetic loci of interest, regardless of the complexity of the polymorphism at these loci, including, for example: (1) simple homozygosity or heterozygosity of a unique locus, as exemplified by DQA or the like; (2) isotypic complexity due to multiple, closely related and closely linked copies of a locus, as exemplified by DRB or the like; and (3) intra-allelic complexity at a locus compounded by interlocus complexity, such as Class I genes or the like. Most known human genetic polymorphisms are of the first, and simplest, type.
Use of the SBT method provides overlapping sequence data comprised of only the copies of the locus of interest as is exemplified by each of the types of HLA loci. The SBT strategy is designed to ensure selection of a given locus with equal representation of each copy of that locus by equal amplification and direct sequencing of mixtures of both alleles of that locus and direct interpretation of the overlapping sequencing patterns generated by this approach. Thus, providing a method for determining genetic polymorphism at one or more genetic loci of interest which can be employed, for example, in HLA typing, detection, evaluation, and/or characterization of genetic diseases such as, for example, sickle cell anemia, cystic fibrosis, Thalassemia, and the like, and detection, evaluation, and/or characterization of polymorphism in genetic loci associated with various cancers such as p53, Ras, myc, associated with carcinomas, leukemias, sarcomas or the like.
Use of the method according to the present invention is exemplified by a system providing for rapid and accurate determination of a major histocompatibility complex class genotype of a subject in a sample (e.g., Class I or Class II). Most particularly, the method is directed to determining at least one HLA Class II gene locus including DRB1, DRB3, DRB4, DRB5, DQB1, DQA1, DPA1 and DPB1 genes. In the case of Class I genotypes, the method is envisioned as being useful to determine A, B, and C loci genes.
To determine a gene locus nucleic acid sequence polymorphism with the method of the present invention, nucleic acid (RNA or DNA) from a sample is isolated. In the case of RNA, cDNA molecules for each allele of at least one gene locus to be sequenced are synthesized by employing a locus-specific oligonucleotide primer that anneals to a conserved region of each allele of each gene locus. According to the present invention, the sample nucleic acid sequence is determined by: amplifying the cDNA molecules or genomic DNA by polymerase chain reaction to generate sufficient product for each allele of each gene locus to be sequenced, with all of the alleles for each gene locus and chromosome to be sequenced being amplified with at least one conserved oligonucleotide primer pair, and at least one of the alleles of each gene locus and chromosome to be sequenced being amplified with at least one non-conserved oligonucleotide primer and at least one conserved primer; preparing the products of each PCR for sequencing (clean); sequencing directly the products of each polymerase chain reaction product to detect each allele at each gene locus of each chromosome, with an enzyme appropriate for DNA sequencing, such as Taq polymerase and a conserved primer specific for each locus that is sequenced; and analyzing each sequenced product for each locus and primer combination(s) to determine the genotype of the subject.
In a preferred embodiment of the present invention the sequence of each polymerase chain reaction product for each allele of each gene locus is determined by analyzing each nucleic acid single and/or overlapping ladder generated for each directly sequenced polymerase chain reaction product. The analysis is conducted by comparing the nucleotide sequence of each allele of each gene locus sequence to known sequences for each locus, followed by comparing the sequence of each gene locus amplified with the non-conserved/conserved oligonucleotide primer pair to the nucleotide sequence of each allele of the gene locus amplified with a conserved oligonucleotide primer pair. Comparison of nucleic acid ladders for sequenced alleles can be conducted visually or using computer software.
In a preferred embodiment, the process of the invention is automated for use in rapid genotype determinations, including diagnosis of genetic diseases. Automation of the process includes isolating the sample nucleic acid with an RNA/DNA extractor; amplifying the synthesized cDNA molecule or the isolated DNA molecule by polymerase chain reaction using a thermocycler to generate the polymerase chain reaction products; sequencing the polymerase chain reaction products in an automated sequencing apparatus; and analyzing each sequenced polymerase chain reaction product with the computer having a database with allelic sequence information and the capacity to conduct the appropriate substraction algorithm for comparing the polymerase chain reaction product sequence for each allele amplified with a conserved oligonucleotide primer pair to the nucleic acid sequence of each allele sequenced with a non-conserved/conserved oligonucleotide primer pair.
The invention further relates to specific groups of oligonucleotide primers useful in the steps of cDNA synthesis, cDNA/genomic DNA amplification by polymerase chain reaction and direct sequencing of the polymerase chain reaction products to determine the nucleotide sequence of each of the alleles at each locus of each chromosome that is amplified. Useful single strand DNA oligonucleotide primers are described in Table 1 herein.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A shows a schematic of the cDNA/PCR/Sequencing experiments for DRB (DRB1, DRB3, DRB4 and DRB5), DQA1, DQB1, DPA1 and DPB1 genes.
FIG. 1B shows a schematic of the primer binding sites on a DRB transcript. Stippled boxes represent primers used in the cDNA synthesis reactions; black boxes represent conserved (or Type 1) primers, used for PRR; checked boxes represent non-conserved (or Type 2) primers, al/so used for PCR; and blank boxes represent sequencing primers.
FIG. 1C shows a schematic of the primer binding sites on a DQB transcript. Stippled boxes represent primers used in the cDNA synthesis reactions; black boxes represent conserved (or Type 1) primers, used for PRR; checked boxes represent non-conserved (or Type 2) primers, also used for PCR; and blank boxes represent sequencing primers.
FIG. 1D shows a schematic of the primer binding sites on a DQA transcript. Stippled boxes represent primers used in the cDNA synthesis reactions; black boxes represent conserved (or Type 1) primers, used for PRR; and blank boxes represent sequencing primers.
FIG. 1E shows a schematic of the primer binding sites on a DPB transcript. Stippled boxes represent primers used in the cDNA synthesis reactions; black boxes represent conserved (or Type 1) primers, used for PRR; and blank boxes represent sequencing primers.
FIG. 1F shows a schematic of the primer binding sites on a DPA transcript. Stippled boxes represent primers used in the cDNA synthesis reactions; black boxes represent conserved (or Type 1) primers, used for PRR; and blank boxes represent sequencing primers.
FIG. 1G shows a schematic of the primer binding sites on DQB1, DRB, DPB1 and DPA1 genes in their germline configuration. Only those primers exclusively used for genomic DNA samples are shown in the Figure.
FIG. 2A shows a flow-chart of the procedure for peripheral blood samples. Each reaction is performed in a different test tube. The reactions are named with capital letter in parenthesis; these letters correspond to those shown in Table II (combinations of primers/reaction). Only the "routine" combinations of primers are shown in this Figure.
FIG. 2B is a flow-chart of the procedure for forensic samples, where DNA is usually the only available genetic material to work with. DNA in these situations is usually isolated from hair, sperm, blood stains, etc. The combinations of primers per reaction shown in the Figure correspond to the "routine" combinations only.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "gene" refers to a segment of DNA, composed of a transcribed region and a regulatory sequence that makes possible a transcription. The term "gene locus" refers to the specific place on the chromosome where a gene is located. The term "allele" refers to the multiple forms of a gene that can exist at a single gene locus at a single chromosome and are distinguishable from the other possible alleles by their differing effects on phenotype (detectable outward manifestations of a specific genotype). "Haplotype" refers to the specific allele composition of the genes at multiple loci on the same chromosome. As used herein the term "genotype" refers to the specific allelic composition of a gene at multiple linked loci at each chromosome (2 haplotypes).
The term "oligonucleotide" as used herein refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three deoxyribonucleotides. The exact number of nucleotides in the molecule will depend on the function of the specific oligonucleotide molecule. As used herein the term "primer" refers to a single stranded DNA oligonucleotide sequence, preferably produced synthetically which is capable of acting as a point of initiation for synthesis of a primer extension product which is complementary to a nucleic acid strand to be copied or a point of initiation for sequencing a DNA molecule. In the case of primers intended for use in synthesizing cDNA or amplifying cDNA or genomic DNA molecules by polymerase chain reaction products, the length and sequence of the primer must be sufficient to prime the synthesis of extension products in the presence of a polymerization enzyme. Preferably, the length of the primer is from about 5-50 nucleotides, more preferably from about 5-20 nucleotides. Specific length and sequence of the primer will depend on complexity of required DNA or RNA target templates, as well as conditions of primer employment such as temperature, ionic strength, and MgCl.sub.2 concentration. When nested primers are used for sequencing, the number of base pairs separating the amplification and sequencing primers on the DNA template are also important considerations.
As used herein, "conserved oligonucleotide primer" (Type 1) refers to an oligonucleotide molecule that corresponds to a region of high DNA sequence conservation (i.e. less than 1-2 nucleotide variations). While the conserved primer need not correspond exactly to the nucleotide template to which it anneals, the conserved primer will have minimal, preferably less than one mismatch with the target nucleotide template. Functionally, conserved primers are capable of equally priming the target nucleotide (cDNA, PCR product, etc.) at high stringency conditions. In contrast to this, as used herein, "non-conserved oligonucleotide primer" (Type 2) refers to an oligonucleotide molecule that has an intended number of mismatches with the possible target nucleotide sequences. The intended number of mismatches can vary with a preferred number of mismatches being about 1-12. Non-conserved primers are characterized by their selective binding to a limited number of alleles at a given locus or at a group of highly homologous loci. The non-conserved primer will bind to the more complementary allele or group of alleles (two or less than two) (i.e., fewer number of mismatches between primer and target template sequence). The specific combinations of conserved and non-conserved primers and the number of reactions per locus or loci used herein are specifically designed to obtain highly accurate results with minimal expenditure of time and cost.
The present invention is directed to a process for determining the sequences of the alleles of polymorphic gene systems carried by any given individual, such as, for example, the human HLA system, genes related to different human genetic disorders, such as sickle cell anemia, cystic fibrosis, or the like, as well as gene systems associated with various cancers, such as p53, myc, or the like. The present invention is exemplified by its utility for determining polymorphism at HLA loci, particularly Class II and Class I genes, the most polymorphic human genetic loci known today, using enzymatic amplification and direct sequencing of the gene cDNA molecules using a limited number of primers and avoiding the use of allele specific oligonucleotides as much as possible. The present method is particularly well suited to determining allelic sequences of Class II HLA genes, thereby providing complete HLA Class II genotype information for a subject. Using the method of the present invention complete Class II HLA typing (DR, DQ and DP) can be performed in about 16 to 24 hours or less.
Generally, the method of the present invention involves: extraction of sample nucleic acid; in the case of RNA, generation of cDNA; cDNA or genomic DNA amplification; direct sequencing of amplification products; and analysis of the direct sequence information. Generation of cDNA, amplifying the cDNA and direct sequencing the cDNA amplification products is accomplished using oligonucleotide primers with specific characteristics, such as those described herein.
A. Oligonucleotide Primers
The oligonucleotide primers of the present invention can be synthesized using any known suitable method, such as phosphotriester and phosphodiester methods. Narang et al., Methods Enzymol., 68:90 (1979); Brown et al., Methods Enzymol., 68:109 (1979). Oligonucleotides can be prepared using a modified solid support such as a Biosearch 8750 DNA synthesizer. Useful primers can also be isolated from a biological source using appropriate restriction endonucleases which cut double stranded DNA at or near a nucleotide sequence of interest for use as a primer.
B. Extraction of Sample Nucleic Acid
In the process of the present invention any source of nucleic acid can be used as the sample nucleic acid, as long as the sample contains the nucleic acid sequence of interest. For example, the sample chosen for the present method can be RNA, DNA or a DNA/RNA hybrid. Typical samples include peripheral blood mononuclear cells, (PBMNC's), lymphoblastoid cell lines (LCL's), hair cells or the like. For determining human HLA Class II and Class I gene polymorphisms LCL's or PBMNC's are preferred. The nucleic acid to be isolated (e.g. RNA or DNA) will depend on the source of genetic material (blood stain, hair, or peripheral blood cells). However, in the case of HLA Class II genes including DRB1-5, DQB1, DQA1, DPA1, DPB1 the preferred isolated nucleic acid is total cellular RNA when the typing is to be done for transplantation purposes or paternity testing. For forensic uses, genomic DNA may be the preferred genetic material in which case different primer considerations would be used. Cytoplasmic and poly(A)+RNA can also be used. It is envisioned that isolation of sample nucleic acid for the present process can be automated using a DNA/RNA extractor (such as Model 341 DNA extractor available from Applied Biosystems, Inc.; Foster City, Calif.).
C. Generation of cDNA
Complementary DNA (cDNA) of the sample nucleic acid is generated using specific oligonucleotide primers and cloned reverse transcriptase following general conditions suggested by the enzyme manufacturer (Bethesda Research Laboratories, Gaithersburg, Md.). Specific differences in type and amount of primers used, dNTP concentrations and elongation times will be readily apparent to those of skill in the art based on the Examples that follow.
D. Polymerase Chain Reaction
Amplification of cDNA or genomic DNA for each gene locus of interest is accomplished using the polymerase chain reaction (PCR) as generally described in U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis. The PCR consists of many repetitions of a cycle which consists of: (a) a denaturation step, which melts both strands of a DNA molecule; (b) an annealing step, which is aimed at allowing the primers to anneal specifically to the melted strands of the DNA molecule; and (c) an extension step, which incorporates to the primers deoxyribonucleotides complementary to those of the strand of DNA to which the primers are annealed. The PCR process, as indicated in the Examples, can be conducted using a Thermocycler (Perkin-Elmer, Cetus, Emeryville, Calif.).
The present invention introduces the use of non-conserved oligonucleotides in the PCR procedure specifically designed to solve the problems associated with, for example, detecting, evaluating, and/or characterizing polymorphism at a polymorphic gene locus or loci of an individual. In the case of HLA typing, the use of non-conserved oligonucleotides addresses the problems one would face in performing HLA typing by sequencing DNA amplified exclusively by using conserved oligonucleotides or allele specific oligonucleotides (see below).
It is understood that the PCR process is designed for the amplification of specific genes with the use of oligonucleotides specific for the particular gene to be amplified. However, even using completely matched primers, in most cases the PCR is not absolutely specific. In the case of HLA typing, for HLA-DRB genes and Class I genes, the use of conserved primers in PCR will generate complex mixtures of templates, which upon direct sequencing will be seen as overlapping sequencing ladders, cumbersome to interpret. Therefore, genes for which the exact nucleotide sequence information is unknown can not be achieved with an adequate level of certainty. Use of non-conserved oligonucleotides which can selectively anneal under high stringency conditions to two or fewer alleles of a gene locus or group of homologous loci can provide sequence information for the different genes at highly homologous loci in complex heterozygote combinations. Thus, the present invention provides a method useful for determining the genotype for polymorphic gene loci. This is of particular importance to HLA typing, and is applicable to Class I HLA typing as well as Class II typing.
The difference between non-conserved primers and allele-specific oligonucleotides resides in that the latter can only be used when the presence of a particular allele is known, and also requires the use of a specific primer for each of the alleles of the polymorphic system. Thus, combining use of a non-conserved primer and conserved primers to amplify the separate alleles of highly homologous polymorphic gene loci can provide simpler DNA polymerase chain reaction product combinations sufficient to allow unambiguous interpretation of direct sequencing ladders of each allele for genotype determinations with moderate expenditure of time and economical cost.
The conditions used for the PCR reactions are preferably the same except for the temperature used in the annealing step, which is different depending on the type of primer used, conserved (Type 1) or non-conserved (Type 2). Reactions that use the former primer type are preferably performed at 37.degree. C. in the annealing step of the cycle, whereas this step is preferably performed at about 55.degree. C. to 60.degree. C. in reactions that use the later type of primers. The concentrations of primers, and buffers used will be apparent from and include the process parameters described in the Examples that follow.
E. Direct Sequencing Of PCR Products
Direct sequencing of double-stranded DNA generated by the PCR is accomplished using an enzyme appropriate to DNA sequencing, such as Taq polymerase, or the like, and specific combinations of reagents at appropriate concentrations. The sequencing procedure can be conducted in an automatic sequencing apparatus such as the 373A Model DNA Sequencer from Applied Biosystems Inc. (Foster City, Calif.). The reagents, including sequencing primers and nucleic acid termination mixtures will be understood by those of skill in the art based on the direct sequencing procedure specified in the following Examples.
F. Analysis Of Direct Sequenced PCR Products
The nucleic acid ladders resulting from direct sequencing the cDNA or genomic DNA for each gene locus of interest can be assessed visually from autoradiograms or by employing a computer programmed with nucleotides sequence information for all alleles of all haplotypes and procedures for comparing sequenced alleles and known alleles of gene loci of interest. In a preferred embodiment of the present invention, the evaluation of gene locus alleles involves a two step process: (a) comparison of the gene sequences of each polymerase chain reaction product (i.e., conserved and non-conserved primer products) with a library of known genotype information such as the information obtained on homologous cell lines very well characterized by methods other than sequencing [Marsh and Bodmer, Immunogenetics, 31:131 (1990)] as well as sequences of individual alleles; followed by (b) comparison of direct sequence information for the polymerase chain reaction product of an allele of a gene locus amplified with a conserved oligonucleotide primer pair and polymerase chain reaction product of alleles of a gene locus or loci amplified with a conserved/non-conserved primer pair. This comparison employs a substitution algorithm or visual cancellation of duplicative sequence ladder information to generate the specific sequence information for each allele of a gene locus.
It is envisioned that the process of the present invention can be used to amplify and sequence known and unknown highly polymorphic systems (e.g., genetic disease-related genes, cancer-related genes, and HLA typing, including Class I, Class II, and Class III HLA typing, and the like). The present process is believed to be useful for paternity testing and forensic medicine, with more accuracy than restriction fragment length polymorphism (RFLP), DNA fingerprinting or dot blot-detection systems. While in the latter only a hybridization pattern is observed, direct sequencing of amplified products shows the exact nucleotide sequence of the amplified genes, and hence is more accurate and reliable.
The method is particularly well suited for Class II HLA typing, reducing its costs, increasing its speed and especially improving its accuracy. As evidenced by the following Examples, sequence polymorphism analysis of DRB1, DRB3, DRB4, DRB5, DQB1, DQA1, DPA1 and DPB1 genes can be rapidly performed in any subject of unknown HLA type by means of enzymatic amplification and direct sequencing of Class II genes using a limited number of conserved and non-conserved oligonucleotides. The approach described herein is entirely automatable using currently available technology and, as opposed to previously described methods using oligonucleotide probes and dot blots, has the advantage of detecting the presence of new allelic sequences or sequence microheterogeneity at the population level. The methodology of the present invention is envisioned to be useful for detailed analyses of the effects of sequence allelism at different Class II HLA loci on graft survival after allogeneic transplantation. The method of the present invention allows rapid and precise sequence analysis of Class II HLA polymorphism in studies of human disease and may be of interest in the search for new Class II sequence variants in large populations of subjects.
The present invention is further described by illustration in the following Examples which are not intended to limit the invention.
EXAMPLE I
1. Preparation of Oligodeoxyribonucleotide Primers and Sequence Primer Combinations Useful for cDNA/PCR/Sequencing Reactions of Class II HLA Genes
All of the oligodeoxyribonucleotide primers described herewithin were synthesized as described below:
Automated Synthesis of oligodeoxyribonucleotide primers: The b-cyanoethylphosphoamidites, obtained from Milligen-Biosearch (Novato, Calif.), were sequentially condensed to a nucleoside derivatized controlled pore glass support using a Biosearch 8750 DNA synthesizer. Condensation cycles included detritylation with dichloroacetic acid in dichloromethane, followed by condensation with benzotriazole and capping with acetic anhydride and 1-methylimidazole in tetrahydrofuran and pyridine, with each cycle time being approximately 9 minutes. Yields at each step were >99% as determined by measuring dimethoxytrityle alcohol release. The methodology for oligodeoxyribonucleotide synthesis is described in Caruthers, et al., Methods Enzymol., 154:287 (1987).
Deprotection and purification of oligodeoxyribonucleotide primers: Deprotection and purification of oligodeoxyribonucleotide primers was performed using the procedure described by Schulhof et al., Nucl. Acids Res., 15:397 (1987). Briefly, the oligodeoxyribonucleotide was removed from the solid support by exposure to concentrated ammonium hydroxide at room temperature for about one hour. The solution containing the partially deprotected oligodeoxyribonucleotide was brought to 65.degree. C. for 16 hours. Ammonia was removed and the residue was subjected to chromatography on a C18 reverse-phase column (RP 304, BioRad, Richmond, Va.) using a linear gradient of 14 to 20% acetonitrile in 0.1 molar ammonium/triethylamine, pH 7.0. The dimethoxytrityle group was removed from the HPLC-purified oligodeoxyribonucleotide by treatment with 70% acetic acid. The detritylated oligodeoxyribonucleotide was recovered by precipitation in ether, vacuum centrifuged until dry, resuspended in water and quantitated by measuring its absorbance at 260 nm.
Using the above procedure, the following oligonucleotide primers corresponding to specified regions of HLA Class II DQA, DQB, DRB, DPB and DPA loci were synthesized (see Table I below) and extensively tested:
2. Combinations of Primers for cDNA/PCR/Sequence Reactions
There are specific combinations of oligonucleotide primers for each reaction and for each locus, including cDNA synthesis, PCR amplification and direct sequencing, which are designed to provide all the necessary sequence information for obtaining highly accurate, fast and inexpensive typing results. These combinations are listed in Table II below as "routine" combinations. In addition, Table II includes a list of "alternative" combinations of oligonucleotides for each locus which may be used to confirm results obtained with the "routine" combinations for a particular locus not expected according to, for instance, known haplotypic maps. These "unexpected" results are usually indicative of the existence of new alleles and/or haplotypes, which can be confirmed with the use of the alternative combinations of oligonucleotides. In any case, each of these combinations of oligonucleotides is characterized by its ability to generate an end-product (sequencing ladder) which is suitable of being accurately read by the naked eye or processed by computer operated under appropriate software.
For typing purposes in the clinical setting, such as in transplantation, the method uses RNA isolated from peripheral blood mononuclear cells as starting material; for forensic purposes, however, DNA is often the only available template. Although for each template (RNA or DNA) different combinations of oligonucleotides are used (see Table II), the general strategy for typing, including the interpretation of the results is essentially the same. The specific combinations of primers for "routine" RNA and DNA analysis, respectively, are described below in more detail. The general overview of the HLA typing strategy is shown in FIGS. 1 and 2.
EXAMPLE II
Protocol: HLA Class II "Typing" by Direct Sequencing of DRB, DQB, DQA, DPA and DPB Genes
1. Cell Lines and Subjects
Lymphoblastoid cell lines (LCLs) representing each of the known Class II haplotypes defined at the 10th International Histocompatibility Workshop [Dupont, Hum. Immunol., 26, 3 (1989)] were provided by Dr. Miriam Segall (University of Minnesota). Forty unrelated subjects who had been previously serologically typed for Class I and Class II antigens were also studied. The serological types of each of the subjects under study were not known to the investigator performing the sequence analysis at the time the analysis was performed. These subjects included both healthy and affected (insulin-dependent diabetes and autoimmune thyroid disease) individuals. The sequenced haplotypes, many in heterozygote combinations, included: DR7 (n=3), DRw17 (n=26), DR4 (n=16), DRw11 (n=8), DRw8 (n=4), DR1 (n=6), DRw15 (n=6), DRw16 (n=2), DRw13 (n=2), DRw14 (n=2), DR212 (n=3), DR5.times.6 (n=3). The cell lines and heterozygote combinations tested are shown in Table III. Since the complexity at DPA and DPB loci is similar to that of DQ genes, the primer combinations for DPA and DPB typing were optimized in a smaller group of homozygote and heterozygote subjects.
2. HLA-DRB, DQB and DQA Transcript Amplification Using Conserved and Non-Conserved Oligonucleotides
Total cellular RNA was prepared from (1 .mu.g) from 5-10.times.10.sup.6 peripheral blood mononuclear cells (PBMNC) or lymphoblastoid cell lines (LCLs) by cesium chloride centrifugation [Chirgwin et al. Biochemistry, 18, 5249 (1979)]. Alternatively, total RNA from peripheral blood (2-10 ml) was partially purified using a much faster protocol [Gouuh, Anal. Biochem., 173, 93 (1988)]. One microgram of total cellular RNA was reverse transcribed with Moloney leukemia virus reverse transcriptase (MLVRT) (200 u, Bethesda Research Laboratories) in 50 mM Tris HCl, pH 8.3, 75 mM KCl, 10 mM DTT, 3mM MgCl.sub.2, in the presence of the ribonuclease inhibitor RNAs in (10 units, Promega), 75 .mu.M each dNTP and 10 pmols of a specific non-sense primer (Table II) in a 20 ml final volume for 30-45 min at 37.degree. C. Eight .mu.l of 10X PCR buffer (500 mM KCl, 100 mM Tris-Cl, pH 8.3, 7.5-15 mMMgCl.sub.2, 0.1% gelatin) were added after the incubation period. A 5'-primer (20 pmols) (Type 1 or Type 2 primers, respectively, see Table II) plus 10 pmols of the non-sense primer and two units of Taq polymerase were also added and the final volume was adjusted to 100 .mu.l with distilled water. The reaction mixture was subjected to 35 cycles of 30 sec at 94.degree. C., 30 sec at 37.degree. C. or 55.degree. C. and 30 sec at 72.degree. C. using a Perkin-Elmer Cetus Thermocycler [see Saiki et al., supra (1985); Mullis and Faloona, supra (1987); Saiki et al., supra (1986); Scharf et al., supra (1986)]. The primers used here, their corresponding sequences and the regions to which they anneal are shown in Table II. The reactions for each locus are usually performed in separate microfuge tubes. However, when using conserved primers, the cDNA and PCR reactions for all loci (DRB, DQA, DQB, DPA and DPB) can be successfully performed simultaneously in the same tube.
3. Direct Sequencing of Amplified Products with Taq Polymerase
The reaction mixture (100 .mu.l ) was freed of unincorporated dNTPs and excess of oligonucleotides by spin-dialysis using Centricon-100 (Amicon) or Ultrafree-100 (millipore) microconcentrators. One half of the retentate (20 .mu.l) was dried down and resuspended in 15 .mu.l of 1X Taq sequencing buffer (50 mM Tris-HCl, pH 9, 10 mM MgCl2). Internal oligonucleotides were used for priming the sequencing of DQB, DRB, DQA, DPB and DPA genes, respectively (Table II). Primers for sequencing each strand are listed in Table II. Only one strand is routinely sequenced for typing; sequencing of the other strand is performed in cases where a new allelic sequence is suspected. Eighty to 100 ng of primer were end-labelled with 10 pmol of gamma-P32 labelled ATP (5000 Ci/mmol, 10 .mu.CI/.mu.L) and 5 units of T4 polynucleotide kinase (Promega Biotec) in a 10 .mu.l final volume. Ten ng of primer (1 .mu.l) were added to the sequencing mixture without extraction of unincorporated labelled ATP, boiled for 5 min., and then left at room temperature for 15 min. Eight units of recombinant Taq polymerase (USB) were added to the mixture. Four .mu.l of the annealed primer/template mixture were later added to 4 .mu.l of each of the stop nucleotide mixes: a) Term mix ddG: 15 microM each dGTP, dATP, dCTP, dTTP; 45 microM ddGTP; b) Term mix ddA: 15 microM each dGTP, dATP, dCTP, dTTP; 600 microM ddATP; c) Term mix ddT: 15 microM each dGTP, dATP, dCTP, dTTP; 1200 microM ddCTP; d) Term mix ddC: 15 microM each dGTP, dATP, dCTP, dTTP; 450 microM ddCTP. The reactions were allowed to proceed for two consecutive periods of 10 min. at 72.degree.-74.degree. C. After the second cycle, each reaction was chased with 2 .mu.l of a 7.5 .mu.M mixture of ATP, GTP, TTP, CTP, and allowed to proceed for 5 min. After spinning down, the reaction was stopped by adding 4 ml of 95% (vol/vol) formamide/20 mM EDTA, heated to 80.degree. C. for 5 min. and loaded on a 0.4 mm thick 6% polyacrylamide/7M urea gel. Electrophoresis was performed at 2500 V for 2 hr, the gel fixed in 5% (vol/vol) glacial acetic acid/5% (v/v) methanol for 15 min, dried, and exposed to Kodak X-Omat film for 4 to 12 hours.
RESULTS
1. Sequence-Based Typing of DR and DQ Polymorphic Genes in Homozygous Typing Cells
Homozygous lymphoblastoid cell lines (LCLs) from the panel of the 10th International Histocompatibility Workshop (Table III) were used as an initial test of the methodology. In total, these cell lines were representative of most of the known DR and DQ alleles at the time the analysis was conducted.
Total cellular RNA isolated from homozygous LCLs was reverse-transcribed and the resultant cDNAs amplified using conserved oligonucleotides specific for DRB1/DRB3/DRB4/DRB5 or DQB1 or DQA1 genes as described in the preceding protocol. The conserved or Type 1 oligonucleotide primers anneal to regions of conserved DNA sequences; these regions are identical among the known alleles at each locus and flank the second exon of Class II genes. These conserved primers, as opposed to non-conserved or Type 2 primers, are designed to amplify all known alleles at DRB, DQA1 and DQB1 loci and, thus, all possible combinations of these alleles in any given heterozygote. The Type 1 oligonucleotides did not cross-amplify templates at loci other than those specified by the oligonucleotides (i.e., the DQA1 primers did not amplify DRB or DQB1 transcripts and vice versa); as expected, the DRB primers also amplified any DRB3, DRB4 or DRB5 transcripts present in addition to DRB1. Sequencing of these amplified templates was performed using a Type 1 primer annealing to a conserved region of the cDNAs internal to the sequence recognized by the amplification primers. FIG. 1A shows the general strategy for the method (SBT) and FIGS. 1B to 1F show the relative position of each of the oligonucleotides used for the cDNA, PCR and sequencing reactions on the mature DRB, DQA and DQB mRNA molecules. The sequences of these primers, the loci they are specific for, the specific positions (codons) to which they anneal and the reaction(s) they are used in are indicated in Table II where the specific combinations of primers that can be used for the cDNA/PCR/sequencing reactions for each locus are identified. As noted in the legend to Table II, some of the primer combinations shown represent alternatives which may be useful in confirming results for a particular locus which do not fit with the expected sequences usually found with the rest of the haplotype. Each cDNA/PCR reaction is usually performed in a separate tube. However, when using Type 1 primers, the cDNA/PCR reactions for all the loci (DRB, DQA, DQB, DPA and DPB) can be performed simultaneously in the same tube. The products of each locus are sequenced in separate tubes. Following the conditions described in the above protocol, the sequence ladders between the sequencing primer and the 5' amplification primer could be clearly read starting from 2 to 14 bases from the sequencing primer binding site. No anomalous amplification products or sequencing ladders were detectable upon direct sequence analysis of amplified DRB, DQB1 and DQA1 cDNAs from the 43 homozygous cell lines tested (Table IIIa). The specific alleles at each Class II HLA locus composing the haplotypes carried by each of these cell lines are shown in Table IIIb. The number of ladders generated for each cell line was always that expected according to the specificity of the amplification primers (one DQB1 and one DQA1 ladder for all cell lines, one DRB ladder for DR1 and DRw8 cell lines and two DRB ladders for haplotypes of the DRw52 and DRw53 supertypic groups). Thus, analysis of the homozygous typing cell lines showed that the Type 1 primers used for cDNA synthesis, PCR and sequencing reactions allowed for accurate amplification and sequencing of all the tested alleles at each of these loci.
2. Amplification and Direct Sequencing of DQA1 and DQB1 cDNAs in Subjects of Unknown HLA Type
DNA sequences have been determined for most HLA Class II allelic specificities defined by conventional HLA typing techniques (March, S. G. E., Bodmer, J. G. HLA-DRB nucleotide sequences, 1990. Immunogenetics 31:141, 1990; Todd, J. A., Bell, J. I., McDefvitt, H. O.: HLA-DQB gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature 329:599, 1987). Comparisons of these sequences indicates that any given DQA1 or DQB1 homozygous or heterozygous allelic combination is characterized by a specific sequencing ladder.
Total RNA from PBMNCs from 40 different subjects was tested to evaluate if the allelic composition of DQA1 and DQB1 homo- and heterozygotes could be determined correctly by direct amplification and sequencing using Type 1 primers. These subjects had been previously serologically typed but the typing information was not known to the investigator who assigned the Class II allelic specificities from the sequencing results. These 40 subjects comprised 27 different heterozygote combinations (Table III). All individuals were assigned DQA1 and DQB1 allelic sequences that were consistent with the serological phenotypes. In all the heterozygotes tested, both allelic sequences could be read clearly from the composite sequence pattern. A unique pattern is found for every particular heterozygote combination in the same way that certain RFLP banding patterns correspond to certain heterozygote allelic combinations. For instance, in a DQB2/DQB1.1 heterozygote one would find the sequence GGGG(A/T)T(T/A)CCGGGC(A/G) at codons 45 to 49 which can only be attributed to that particular allele combination. In practice, interpretation of heterozygous sequence ladders is initiated by reading certain polymorphic positions where allele-specific bases may be found, such as, for instance, the second base of codon 46, where DQB1*0201 is the only allele that has an A. The sequences of the two possible templates are then deduced and compared with the sequences of all known alleles at the different loci.
The absence of expected bands or the presence of unexpected bands for a particular allele or allelic combination is therefore suggestive of sequence heterogeneity, i.e., new alleles. The same can be said for DPA1 and DPB1 typing when appropriate primer combinations are used (Table III). For instance, substitution of the A at the second base of codon 46 would strongly suggest the presence of a sequence variant of DQB1*0201. Once detected, the sequence of the variant can be confirmed after selective amplification of the variant or by subcloning the amplified products.
3. Amplification and Direct Sequencing of DRB cDNAs From Subjects of Unknown HLA Type
As described above, the use of Type 1 primers allows the unambiguous sequencing of all heterozygous combinations of DQA1 and DQB1 alleles. The same can be said for DPA1 and DPB1 typing when appropriate primer combinations are used (Table II). Because of the isotypic complexity of DRB genes (expression of more than one DRB locus by certain haplotypes), amplification and sequencing of cDNAs from DRB heterozygotes with Type 1 primers can generate up to four overlapping ladders, thus generating complex sequencing patterns.
DRB cDNAs from the same 40 individual tested above for DQA1 and DQB1 genes were amplified and sequenced using DRB-specific Type 1 primers. As mentioned above, these 40 individuals comprised 27 different heterozygote combinations, including several examples from each of the groups of complex DRB allelic combinations which would generate up to four sequencing ladders. The DRB sequence ladders generated with Type 1 primers were analyzed as described above for DQA1 and DQB1 loci: highly polymorphic positions were analyzed first for the presence of bands unique to specific alleles or groups of alleles (i.e., DR4) and the sequences deduced and compared with the sequences of all known alleles at all loci. For all but one sample, the information deduced from these sequencing experiments matched the independently determined serological phenotypes of the subject under study as well as the DQA1 and DQB1 allelic types assigned to these individuals by direct sequencing of these genes as described above. The inconsistent sample had been serologically typed as DRw13/DR4 but was typed by sequence analysis as DRw13/DRw8-Dw8.1. The presence of a DRB1*0801 allele instead of a DRB1*0401 allele was confirmed in a repeated experiment; we thus believe that the serological typing was in error. In all the 40 cases, all DQB1, DQA1 and DRB1 templates had been equally amplified and sequenced with a similar efficiency by the use of Type 1 primers. DRB3, DRB4 and DRB5 sequence ladders could be read in all but one case (a DRB3*0101 [DRw52a] sequence was not initially observed in a DRw13/DRw17 heterozygote). Since DRB3*0101 is in linkage disequilibrium with DRB1*0301, the former allele was expected to be found in the overlapping ladder as well. In order to rule out the possibility of an error, the investigator assigning the HLA types from the sequencing ladders repeated the typing of this individual; the DRB3*0101 could be read in the repeated experiment.
Although the results generated by the use of Type 1 primers were compatible with the serological phenotypes, the exclusive use of Type 1 primers will not allow in all cases to assign each of the specific ladders to each of the expressed loci in all possible heterozygotes. Given below are the most complex situations which cannot be addressed by the exclusive use of Type 1 primers: 1) distinction among the different DR4 allelic sequences in certain heterozygotes since they differ by only a few nucleotide base pairs and such differences could be masked by the presence of additional ladders; 2) to distinguish between DRB1*1601 and DRB1*1502 since their sequence differences will be masked by those of their linked DRB5 alleles; 3) to distinguish between DRB1*1301 and DRB1*1302 (which only differ at codon 86 since this difference can also be masked by other ladders; and finally 4) distinction between DRB1*0301 and DRB1*0302 in specific heterozygote combinations.
We have thus developed a more informative strategy to deal with DRB; this strategy, which consists of the additional use of non-conserved (Type 2) primers permits the clear elucidation of even the most complex combination of the four DRB sequences that might be present in an individual. These non-conserved primers, as opposed to allele-specific primers, are designed to be used in reactions performed simultaneously with the reactions using Type 1 primers and aim at selectively amplifying certain ladders from the complex sequencing patterns without requiring previous typing information.
Analysis of the sequence variability of the second exon of the DRB genes has allowed us to identify two regions which could be used to design non-conserved (Type 2) primers: 1) codons 5-13; and 2) codons 29-35. The sequence of the former region follows a group-specific sequence pattern, i.e., a sequence shared by groups of alleles at individual loci. The later region exhibits a scattered nucleotide polymorphism in DRB1 and DRB3, DRB4 and DRB5 genes. We designed five different non-conserved primers annealing to these two polymorphic regions: 1) DRB23 (specific for DR2-DRB1 ladders); 2) DRB24 (specific for DRw17-, DRw18-, DRw13-, DRw14-, DRw11-, DRw12-, and DRw8- DRB1 ladders); 3) DRB25 (specific for DR4- DRB1 ladders); 4) DRB16 and 5) DRB17, the latter two primers annealing to the second region of moderate polymorphism (from 1 to 5 nucleotides different among the known alleles for each locus) (Tables I and IV). Because of the different nature and distribution of mismatches between these primers and the different DRB templates, the type of templates amplified selectively by these primers will be different. Each Of the first three primers will amplify up to two DRB1 cDNAs in any given heterozygote and will not amplify any DRB3, DRB4 or DRB5 cDNAs. On the contrary, the use of primers DRB16 and DRB17 will allow the random selective amplification of certain transcripts from DRB1, DRB3, DRB4 and/or DRB5 loci in most heterozygote combinations. We therefore tested these primers in order to determine which combination would give the best discriminatory results for DRB typing. Furthermore, since the sequences of these primers carry from 0-12 mismatches with the sequences of the known DRB alleles at the different DRB loci, their use allowed us to determine the number of mismatches between the primers and each of the possible cDNAs that are required to obtain such selective amplification of DRB transcripts. The specific combinations of primers used for the cDNA/PCR/sequencing reactions are shown in Table II above. The results of this analysis are shown below.
These primers were able to selectively amplify certain DRB templates in all the heterozygous combinations tested. In heterozygotes carrying the alleles these primers are matched with, these alleles were selectively amplified; in heterozygotes not carrying the alleles specifically recognized by the primers, the DRB templates which had the fewest base pair mismatches with the primers were selectively amplified in the PCR. Specific examples of the latter are shown in Table V. As shown in Table V, Type 2 primers could differentially amplify DRB transcripts from the combinations of allelic cDNAs that differ from each other in as few as one nucleotide substitution, provided that high stringency annealing conditions are used for the PCR (annealing at 55.degree. C.). For example, in the heterozygote combination DRw13/DRw8-Dw8.1, the DRB1*0801 allele (3 mismatches with the primer) was selected over DRB1*1301 and DRB3*0101 genes (each has 4 mismatches with the primer) by the DRB16 oligonucleotide primer. Although DRB3*0101 or DRB3*0201 and DRB5*0101 genes all harbour one mismatch with primer DRB17, this oligonucleotide selected the DRB5*0101 sequence in a DRw11/DRw15 heterozygote (Table V). It is possible that the differential positioning of the mismatches within the sequence recognized by the oligonucleotide also has an influence on the stability of the primer/cDNA complex and hence on the outcome of the PCR.
The ability of non-conserved primers to select certain alleles in heterozygote combinations was also tested for DQB1 genes (Table V). As with DRB-specific Type 2 primers, the use of high temperatures (55.degree. C.) in the annealing step of the PCR was required for achieving the selective amplification of single DQB1 alleles in heterozygotes with non-conserved primers. For instance, when annealing of primer DQB6 was allowed to proceed at 37.degree. C. in cDNAs from a DQB1*0301/DQB1*0501 and a DQB1*0201/DQB1*0603 heterozygote, both alleles in both heterozygotes were equally amplified. At 55.degree. C., the allele with the most homologous sequence to the 5' primer, was amplified over the other in the PCR. Combinations of alleles both differing from the primer in two nucleotides but in different relative position were also differentially amplified with a non-conserved primer. For instance, primer DQB6 selected the DQB1*0604 sequence in a DQB1*0604/DQB1*0502 heterozygote (Table V). Five nucleotides separate the two mismatches between the DQB1*0604 allele and the DQB6 primer, whereas only two nucleotides separate the mismatches between the DQB1*0502 and the primer.
These results clearly indicate that the oligonucleotide primers annealing to polymorphic regions at the 5' end of the target cDNAs can be tailored to achieve a reproducible selective amplification of a limited number of DRB or DQB templates in complex heterozygous combinations. Although the use of Type 1 primers allows the unambiguous sequencing of all possible DQA, DQB, DPA and DPB heterozygotes, such an approach will not give absolute discriminatory information for all DRB heterozygotes. We have shown that the simultaneous use of Type 1 and Type 2 primers for DRB will permit the clear elucidation of even the most complex of all DRB heterozygote combinations. When DRB-SBT is used for typing purposes, we perform three Type 2-reactions (using DRB23, 24 and 25) simultaneously with a Type 1-reaction (Table II). The simultaneous use of these reactions using these primers has the highest discriminatory power for complete DRB typing in a single run and allows the identification of novel sequence heterogeneity. Only one Type 1 reaction is required for DQB1, one for DQA1, one for DPA1 and one for DPB1 (Table II).
EXAMPLE III
Determining Unknown HLA Type of Subjects by Direct Sequencing of the Second Exon of Class II Genes
Routine HLA typing of large populations of individuals for sequence polymorphisms can be performed by the use of the methodology reported here which can also identify previously unknown allelic variants. FIGS. 2A and 2B show a flow-chart for the-routine protocol used to determine sequence allelism of individuals of unknown HLA types.
1. Employment of Primer Combinations for cDNA, PCR and Direct Sequencing Using RNA as Initial Template
For synthesizing cDNA molecules, the present invention provides single strand DNA anti-sense oligonucleotide primers that anneal to conserved regions of the gene mRNAs to be reverse transcribed, amplified and sequenced. These oligonucleotide primers include an oligonucleotide sequence that: (1) anneals to a conserved region (codons 105 through 111) shared by all the alleles at all the DRB loci, the latter being DRB1, DRB3, DRB4 and DRB5, respectively (e.g., primer DRB20). Four simultaneous cDNA reactions (one per tube) are performed for DRB typing, all using primer DRB20 (reactions A, B, C and D in Table II and FIG. 2A); (2) anneals to a conserved region (codons 105 through 111) shared by all the alleles at the DQB locus (e.g. primer DQB7) (reaction E in Table II and FIG. 2A); (3) anneals to a conserved region (codons 147 through 157) shared by all the alleles at the DQA locus (e.g. primer DQA9) (reaction F in Table II and FIG. 2A); (4) anneals to a conserved region (codons 105 through 111) shared by all the alleles at the DPB locus (e.g. primer DPB11) (reaction G in Table II and FIG. 2A); (5) anneals to a conserved region (codons 104 through 110) shared by all the alleles at the DPA locus (e.g. primer DPA14) (reaction H in Table II and FIG. 2A); (6) anneals to a conserved region (codons 222 through 228) shared by all the alleles at the DPA locus (e.g. primer DPA19) (reaction I in Table II and FIG. 2A). The specific oligonucleotides added to each of these reactions once the cDNA synthesis is done in order to amplify and sequence the products are indicated below as well as in Table II and in FIG. 2.
To amplify cDNA molecules corresponding to each expressed DRB loci of each chromosome (DRB1 and DRB3 or DRB4 or DRB5, depending on the haplotype--isotypic complexity--), a conserved oligonucleotide primer which anneals to codons -32 to -26 (e.g. oligonucleotide DRB11) is added to one of the four tubes where the cDNA synthesis reactions corresponding to DRB genes took place. The combination of the cDNA synthesis reaction primer and the newly added conserved primer is used to amplify all the alleles at all DRB loci expressed by a given individual. Each of the remaining three tubes containing DRB cDNA products receives one of three different non-conserved oligonucleotides (also called Type 2) annealing to codons 7-13 (e.g. primer DRB23), 5-11 (e.g. primer DRB24), 6-13 (e.g. primer DRB25), respectively. Each non-conserved primer is designed to favor the amplification of cDNAs corresponding to different groups of alleles at the DRB1 locus. Comparison of the sequencing ladders generated by these four reactions allows complete and accurate interpretation of the sequences corresponding to each of the four possible DRB genes expressed by a given individual (one or two for each of the parental chromosomes).
For the DQB1 locus, a conserved oligonucleotide primer which anneals to codons 1-7 of the DQB cDNAs (e.g. primer DQB13) can be used for amplifying each of the DQB1 genes expressed in any given individual (one for each parental chromosome). In the case of the DQA1 locus, a conserved single strand DNA oligonucleotide primer useful for amplifying each of the DQA1 genes expressed in any given subject anneals to codons -10 to -4 of the DQA1 cDNA (e.g. primer DQA10). For the DPB1 locus, a conserved oligonucleotide (e.g. primer DPB10) annealing to codons -19 to -13, is used to amplify each of the expressed DPB1 genes in any given subject. For DPA1 locus, a conserved oligonucleotide (e.g. primer DPA15) annealing to codons -23 to -17, is used to amplify each of the expressed DPA1 genes in a given subject. In a separate reaction, conserved primer DPA18, annealing to codons 59-65 of the DPA1 cDNAs is used in combination with the cDNA primer DPA19 to amplify each of the expressed DPA1 genes in any individual. This second DPA1 reaction is targeted at a second polymorphic region of this gene.
Primers useful in direct sequencing the polymerase chain reaction products corresponding to DRB loci include an anti-sense oligonucleotide primer (e.g. DRB12) annealing to codons 87-94 of all the alleles at DRB loci; this primer is used for sequencing the products generated by the first of the four DRB reactions. For direct sequencing the polymerase chain reaction products generated with the other three DRB reactions, an anti-sense oligonucleotide annealing to codons 97-103 of all the alleles at DRB1 locus can be used (e.g. primer DRB30). The use of a different sequencing oligonucleotide in these three DRB reactions allows reading of downstream polymorphic regions of DRB1 genes not seen in the first DRB reaction which uses the example sequencing primer DRB12. Primers useful in direct sequencing the polymerase chain reaction products corresponding to DQB1 locus include an anti-sense oligonucleotide primer (e.g. DQB5) annealing to codons 78-83 of all the alleles at this locus. Direct sequencing of polymerase chain reaction products corresponding to DQA1 locus include an anti-sense oligonucleotide primer (e.g. DQA29) annealing to codons 88-95 of all the alleles at this locus. Direct sequencing of polymerase chain reaction products corresponding to DPB1 locus include a sense oligonucleotide primer (e.g. DPB13) annealing to codons 12/-5 of all the alleles at this locus. For direct sequencing of polymerase chain reaction products for the DPA1 reaction which used primers DPA14 and DPA15, an anti-sense oligonucleotide annealing to codons 88-94 of all the alleles at this locus can be used (e.g. primer DPA16). For direct sequencing of polymerase chain reaction products for the DPA1 reaction which used primers DPA19 and DPA18, an anti-sense oligonucleotide annealing to codons 214-220 of all the alleles at this locus can be used (e.g. primer DPA20).
2. Employment of Primer Combinations for PCR and Direct Sequencing Using DNA Templates
To amplify DNA molecules corresponding to each DRB loci of each chromosome a conserved anti-sense oligonucleotide primer annealing to base pairs 18-38 of intron 3 (e.g. oligonucleotide DRB1406) is added to each of four PCR reaction tubes (reactions S, V, T and U in Table II and FIG. 2B). Each of these four tubes will receive a different additional oligonucleotide annealing to codons -4 to +3 (e.g. primer DRB22), to codons 7-13 (e.g. primer DRB23), 5-11 (e.g. primer DRB24), 6-13 (e.g. primer DRB25), respectively. The first reaction is used to amplify all the alleles at all DRB loci carried by a given individual. Each of the remaining three reactions is designed to favor the amplification of DNA corresponding to different groups of alleles at the DRB1 locus. As with RNA templates, comparison of the sequencing ladders generated by these four reactions allows complete and accurate interpretation of the sequences corresponding to each of the four possible DRB genes expressed by a given individual (one or two for each of the parental chromosomes).
For the DQB1 locus, two conserved oligonucleotide primers which anneal to codons 88-94 (e.g. primer DQB932) and 11-17 (e.g. primer DQB931) or 1-7 (e.g. primer DQB13) can be used for amplifying each of the DQB1 genes carried by any given individual (one for each parental chromosome) (reaction W in Table II and FIG. 2B). For the DPB1 locus, two conserved oligonucleotides (a primer, e.g. DPB14, annealing to base pairs -42 to -62 of intron 2, and a primer e.g. DPB15, annealing base pairs 39-59 of intron 3) are used to amplify each of the DPB1 genes carried by any given subject (reaction X in Table II and FIG. 2B). For DPA1 locus a conserved oligonucleotide such as DPA10 (annealing to base pairs -69 to -50 of intron 2) and DPA11 (annealing to base pairs 55-71 of intron 3) are used to amplify each of the DPA1 genes carried by a given subject (reaction Y in Table II and FIG. 2B).
Primers useful in direct sequencing the polymerase chain reaction products generated from DNA templates corresponding to DRB loci include an anti-sense oligonucleotide primer (e.g. DRB12) annealing to codons 87-94 of all alleles at DRB loci; this primer is used for sequencing the products generated by the first of the four DRB reactions. For direct sequencing the polymerase chain reaction products generated with the other three DRB reactions, a sense oligonucleotide annealing to codons 39-46 of all the alleles at DRB1 locus can be used (e.g. primer DRB1400). The use of a different sequencing oligonucleotide in these three DRB reactions allows reading of downstream polymorphic regions of DRB1 genes not seen in the first DRB reaction which uses the example sequencing primer DRB12. Primers useful in direct sequencing the polymerase chain reaction products corresponding to DQB1 locus include an anti-sense oligonucleotide primer (e.g. DQB5) annealing to codons 78-83 of all the alleles at this locus. Direct sequencing of polymerase chain reaction products corresponding to DPB1 locus include an anti-sense oligonucleotide primer (e.g. DPB16) annealing to base pairs 1-21 of intron 3 of all the alleles at this locus. For direct sequencing of polymerase chain reaction products for the DPA1 reaction an anti-sense oligonucleotide annealing to codons 76-82 of all the alleles at this locus can be used (e.g. primer DPA12).
Procedure for Determining Unknown HLA Type
A subject of unknown HLA type, diseased or not, is to be typed for Class II HLA polymorphism. From 10 to 50 mL of peripheral blood are drawn. The peripheral blood mononuclear cells are prepared by centrifugation over Ficoll-Hypaque gradients. The cells are then lysed in guanidium isothyocianate and total cellular RNA prepared using conventional methods (either by centrifugation on cesium chloride gradients, which lasts about 16 hours, or by the guanidium isothyocianate-phenol-chlorophorm extraction method, which can be performed in less than 4 hours. See Gouuh, supra (1988); Johns et al., Anal. Biochem., 180:276 (1989). Otherwise genomic DNA from these cells or other sources (hair, blood stains, sperm, etc.) can be prepared with conventional methods such as provided by Higuchi, R. in PCR Technology, Erlich, M. (ed.), Stockton Press:31 (1989). DQB1, DQA1, DRB (DRB1, DRB3/4/5), DPA1 and DPB1 cDNA molecules are synthesized from total RNA using locus-specific primers. Approximately, one microgram of RNA is reverse transcribed with MoLVRT (reverse transcriptase) and DRB (CODRB20), DQB (CODQB7), DQA (CODQA9), DPB (DPB11) and DPA (DPA14, DPA19) (optional)--specific non-sense primers in a 20 uL final volume reaction (30-60 minute incubation). The reaction for each Class II gene is performed in a different tube, but they can be performed in the same tube if preferred. For routine purposes, four simultaneous reactions are performed for DRB, one for DQB, one for DQA1, one for DPB1, and two for DPA1 gene products.
Once these reactions are completed, the enzymatic amplification of the respective cDNA molecules is then performed by directly adding to the 20 uL reverse transcription reaction, the reagents needed for the amplification step. Alternatively, if DNA is used, the primer combinations used for the PCR are those shown in Table II herein (the anti-sense primers as well as the sense primers will be different). This includes the PCR reagents and appropriate conserved and non-conserved oligonucleotide primers. This example uses four reactions for DRB (tubes 1, 2, 3 and 4), one for DQB (tube 5), one for DQA (tube 6), one for DPB (tube 7), and two for DPA (tubes 8 and 9, respectively). Reactions 2, 3 and 4 incorporate primers DRB23, DRB24 and DRB25, respectively. For rapid typing (in less than 24 hours), the latter are the preferred combinations. Alternative combinations of the primers that can be used are shown in Table II.
Once completed, the reactions are spun-dialyzed for about 15 minutes using Centricon (Amicon, Ultrafree (milli-pore)) or similar columns to remove unincorporated primers and dNTPs. The retentate or one half of the recovered retentate for each reaction is then directly sequenced using Taq polymerase and the primers described in Table II for each combination of primers used in the cDNA/PCR reactions using P-32 end-labeled (10 minutes) locus-specific sequencing primers (35 minutes).
The sequencing reactions products are loaded on an acrylmide gel, electrophoresed in 2-3 hours and exposed to X-ray films for 4-12 hours. The gels are read and results from gels are compared to nucleotide sequences corresponding to all possible alleles.
Comparisons can be made visually using the naked eye or using a personal computer and a software package including the nucleotide sequences of all alleles of all haplotypes and routines which indicate how the comparison is to be performed as well as subroutines which will allow identification of new allelic sequences.
Applicants state that the paper copy of the above "Sequence Listing" Section of the present application, and the computer readable form of the same submitted therewith, are the same.
Claims
What is claimed:
1. A method for determining the genotype of one or more HLA Class H polymorphic gene loci of interest selected from the group consisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and --DPA1, of n subject in a sample containing subject RNA using a combination of conserved and non-conserved oligonucleotide primers, said method comprising: (a) synthesizing in separate reactions, from RNA isolated from said sample, a cDNA molecule for each allele of each gene locus of interest, using a conserved oligonucleotide antisense primer that anneals to a nucleotide region conserved between each allele of each gene loci to be genotyped; (b) amplifying said cDNA by at least one amplifying reaction to generate sufficient first and second double stranded amplifying reaction products for sequencing, using in a separate first amplifying reaction for each of said gene loci of interest encoded by the 3'-end of the alpha 1 domain exon, an oligonucleotide primer pair comprising a first conserved primer and a second conserved primer or, when said gene locus is HLA-DQB or HLA-DRB, optionally a first nonconserved primer for each of said gene loci of interest, wherein one of said first and second conserved oligonucleotide primers anneals to at least one allele of each of said loci of interest; (c) sequencing directly at least one strand of said amplifying reaction product of each of said gene loci of interest to produce at least one sequencing ladder for each of said gene loci of interest, using in each sequencing reaction a third conserved oligonucleotide primer for each of said gene loci of interest; (d) comparing the nucleotide sequence obtained from each sequencing ladder to known sequences for each such gene locus; and, optionally when said gene locus is HLA-DQB or HLA-DRB, (e) comparing the nucleotide sequence of each sequencing ladder optionally obtained with a conserved oligonucleotide primer pair to the nucleotide sequence of each allele of such gene locus amplified with a conserved/nonconserved oligonucleotide primer pair, wherein said gene loci of interest are one or more gene loci whereby the Zenotype of said HLA Class II gene locus is obtained, wherein when said gene locus is HLA-DQA1 (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 3; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 3 and SEQ. ID. NO: 7: and (c) said third conserved primer is depicted in SEQ. ID. NO: 14 or SEQ. ID. NO: 15; wherein when said gene locus is HLA:DOB1 said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of (1) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 1: (b) said first and second conserved primers are depicted in SEQ. ID. NO: 1 and SEQ. ID. NO: 4; and (c) said third conserved primer is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12; (2) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 1: (b) said tint conserved primer is depicted in SEQ. ID. NO: 1 and said first nonconserved primer is depicted SEQ. ID. NO: 47 and wherein annealing of said conserved/nonconserved primer pair occurs at about 37.degree. C.; and (c) said third conserved primer is depicted in SEQ. ID. NO: 4; (3) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 1: (b) said first conserved primer is depicted in SEQ. ID. NO: 1 and said first nonconserved primer is depicted SEQ. ID. NO: 47 and wherein annealing of said conserved/nonconserved primer pair occurs at about 55.degree. C.-60.degree. C.; and (c) said third conserved primer is depicted in SEQ. ID. NO: 10or SEQ. ID. NO: 12; (4) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 1: (b) said first conserved primer is depicted in SEQ. ID. NO: 1 and said first nonconserved primer is depicted SEQ. ID. NO: 48; and (c) said third conserved primer is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12; and (5) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 1: (b) said first conserved primer is depicted in SEQ. ID. NO: 1 and said first nonconserved primer is depicted SEQ. ID. NO: 49: and (c) said third conserved primer is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12; wherein when said gene locus is HLA-DPB1, said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of (1) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 24; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 24 and SEQ. ID. NO: 23: and (c) said third conserved primer is depicted in SEQ. ID. NO: 25 or SEQ. ID. NO: 26: and (2) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 25; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 25 and SEQ. ID. NO: 23: and (c) said third conserved primer is depicted in SEQ. ID. NO: 26; wherein when said gene locus is HLA-DPA1, said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of (1) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 31; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 31 and SEQ. ID. NO: 32; and (c) said third conserved primer is depicted in SEQ. ID. NO: 33 or SEQ. ID. NO: 34; (2) (a) said conserved oligonucleotide and sense primer is depicted in SEQ. ID. NO: 39; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 39 and SEQ. ID. NO: 38: and (c) said third conserved primer is depicted in SEQ. ID. NO: 40 or SEQ. ID. NO: 41; (3) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 33; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 33 and SEQ. ID. NO: 32; and (c) said third conserved primer is depicted in SEQ. ID. NO: 34; wherein when said gene locus is HLA-DRB, said combination of conserved and non-conserved oligonucleotide primers is selected from the group consisting of (1) (a) said conserved oligonucleotide and sense primer is depicted in SEQ. ID. NO: 2: (b) said first and second conserved primers are depicted in SEQ. ID. NO: 2 and SEQ. ID. NO: 6: and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; (2) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said first conserved primer is depicted in SEQ. ID. NO: 2 and said first nonconserved primer is depicted in SEQ. ID, NO: 42 and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; (3) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said first conserved primer is depicted in SEQ. ID. NO: 2 and said first nonconserved primer is depicted in SEQ, ID. NO: 43; and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ, ID. NO: 13; (4) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said first conserved primer is depicted in SEQ, ID. NO: 2 and said first nonconserved primer is depicted in SEQ. ID, NO: 44: and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; (5) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said amplifying has four discrete amplifying steps, and wherein in first discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 2 and said second conserved primer is depicted in SEQ. ID. NO: 6: and in a second discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 2 and said first nonconserved primer is depicted in SEQ. ID. NO: 42), and in a third discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 2 and said second nonconserved primer is depicted in SEQ. ID. NO: 43 and in a fourth discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 2 and said third nonconserved primer is depicted in SEQ. ID. NO: 44; and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13 or SEQ. ID. NO: 5; (6) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said first conserved primer is depicted in SEQ. ID. NO: 2 and said first nonconserved primer is depicted in SEQ. ID. NO: 45; and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; (7) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said first conserved primer is depicted in SEQ. ID. NO: 2 and said first nonconserved primer is depicted in SEQ. ID. NO: 46: and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13; and (8) (a) said conserved oligonucleotide antisense primer is depicted in SEQ. ID. NO: 2; (b) said first and second conserved primers are depicted in SEQ. ID. NO: 2 and SEQ. ID. NO: 5; and (c) said third conserved primer is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
2. A method for determining the genotype of one of more HLA Class II polymorphic gene loci of interest selected from the group consisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and DPA1 of a subject in a sample containing subject genomic DNA using a combination of conserved and nonconserved oligonucleotide primers, said method comprising; (a) amplifying said genomic DNA by at least one amplifying reaction to generate sufficient tint and second double stranded amplifying reaction products for sequencing, using in a separate first amplifying reaction for each of said gene loci of interest encoded by the 3'-end of the alpha 1 domain exon, an oligonucleotide primer pair comprising a first conserved primer and either a second conserved primer or a first nonconserved primer for each of said gene loci of interest, wherein said conserved primers anneal to a nucleotide sequence conserved between each allele of each gene loci to be genotyped, and wherein said nonconserved primer anneals to a nucleotide sequence of a group of two or less alleles of each gene loci to be genotyped; and (b) sequencing directly at least one strand of said amplifying reaction product for each of said gene loci of interest to produce at least one sequencing ladder for each of said gene loci of interest, using in each sequencing reaction a third conserved oligonucleotide primer for each of raid gene loci of interest whereby a nucleotide sequence is obtained for each sequencing ladder; (c) comparing said nucleotide sequence obtained from each sequencing ladder to known sequences for each gene locus of interest; and (d) when a first non-conserved primer is used, comparing said nucleotide sequence of each sequencing ladder obtained with a conserved oligonucleotide primer pair to the nucleotide sequence of each allele of such gene locus amplified with a conserved/nonconserved oligonucleotide primer pair, whereby the genotype of one or more HLA Class II gene loci is obtained, wherein when said gene locus is HLA-DQB-1 (a) said first and second conserved primers are depicted in SEQ. ID. NO: 8and SEQ. ID. NO: 9; and (b) said third conserved primer is depicted in SEQ. ID. NO: 12; wherein when said gene locus is HLA-DPB1, said combination of conserved and non-conserved primers are selected from the group consisting of (1) (a) said first and second conserved primers are depicted in SEQ. ID. NO: 27 and SEQ. ID. NO: 28; and (b) said third conserved primer is depicted in SEQ. ID. NO: 29 or SEQ. ID. NO: 30; and (2) (a) said first and second conserved primers are depicted in SEQ. ID. NO: 27 and SEQ. ID. NO: 29; and (b) said third conserved primer is depicted n SEQ. ID. NO: 30; wherein when said one locus is HLA-DPA1, (a) said first and second conserved primers are depicted in SEQ. ID. NO: 35 and SEQ. ID. NO: 36; and (b) said third conserved primer is depicted in SEQ. ID. NO: 37; wherein when said gene locus is HLA-DRB, said combination of conserved and non-conserved primers are selected from the group consisting of (1) (a) said first and second conserved primers are depicted in SEQ. ID. NO: 20 and said first nonconserved primer is depicted in SEQ. ID. NO: 5; and (b) said third conserved primer is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16; (2) (a) said first conserved primer is depicted in SEQ. ID. NO: 20 and said first nonconserved primer is depicted in SEQ. ID. 43; and (b) said third conserved primer is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16; (3) (a) said first conserved primer is depicted in SEQ. ID. NO: 20 and said first nonconserved primer is depicted in SEQ. ID. NO: 44; and (b) said third conserved primer is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16; (4) (a) said first conserved primer is depicted in SEQ. ID. NO: 20 and said first nonconserved primer is depicted in SEQ. ID. NO: 42; and (b) said third conserved primer is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16; (5) (a) said amplifying has four discrete amplifying steps and wherein in a first discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 20 and said second conserved primer is depicted in SEQ. ID. NO: 5, and in a second discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 20 and said first nonconserved primer is depicted in SEQ. ID. NO: 43, and in a third discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 20 and said second nonconserved primer is depicted in SEQ. ED. NO: 44 and in a fourth discrete amplifying step said first conserved primer is depicted in SEQ. ID. NO: 20 and said third nonconserved primer is depicted in SEQ. ID. NO: 42; and (b) said third conserved primer is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16; (6) (a) said first and second conserved primers are depicted in SEQ. ID. NO: 13 and SEQ. ID. NO: 22; and (b) said third conserved primer is depicted in SEQ. ID. NO: 21; (7) (a) said first conserved prime is depicted in SEQ. ID. NO: 17 and said nonconserved primer is depicted in SEQ. ID. NO: 18; and (b) said third conserved primer is depicted in SEQ. ID. NO: 19; (8) (a) said first conserved primer is depicted in SEQ. ID. NO: 20 and said nonconserved primer is depicted in SEQ. ID. NO: 45; and (b) said third conserved primer is depicted in SEQ. ID. NO: 21 or SEQ. ID. NO: 13; and (9) (a) said first conserved primer is depicted in SEQ. ID. NO: 20 and said nonconserved primer is depicted in SEQ. ID. NO: 46; and (b) said third conserved primer is depicted in SEQ. ID. NO: 21 or SEQ. ID. NO: 13.
3. A method for determining the genotype of one or more HLA Class II polymorphic gene loci of interest selected from the group consisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, --DPB1 and DPA1, of a subject in a sample containing subject RNA using a combination of conserved and nonconserved oligonucleotide primers, said method comprising: (a) synthesizing in separate reactions, from RNA isolated from said sample, a cDNA molecule for each allele of each gene locus of interest, using a conserved oligonucleotide antisense primer for each of said gene loci of interest; (b) amplifying said cDNA by at least one amplifying reaction to generate sufficient first and second double stranded amplifying reaction products for sequencing, using in a separate first amplifying reaction of each of said gene loci of interest encoded by the 3'-end of the alpha 1 domain exon, an oligonucleotide primer pair comprising a first conserved primer and either a second conserved primer or a first nonconserved primer for each of said gene loci of interest, wherein one of said first and second conserved oligonucleotide primers anneals to at least one allele of each of said loci of interest; (c) sequencing directly at least one strand of said amplifying reaction product for each of said gene loci of interest to produce at least one sequencing ladder for each of said gene loci of interest, using in each sequencing reaction a third conserved oligonucleotide primer for each of said gene loci of interest; (d) comparing the nucleotide sequences obtained from each sequencing ladder to known sequences for each such gene locus; (e) when a first non-conserved primer is used, comparing the nucleotide sequence of each sequencing ladder obtained with a conserved oligonucleotide primer pair to the nucleotide sequence of each allele of such gene locus amplified with a conserved/nonconserved oligonucleotide primer pair, wherein said gene loci of interest are one or more gene loci whereby the genotype of said HLA Class II gene locus is obtained: (f) wherein: i) said conserved oligonucleotide antisense palmer of HLA-DQA1 is depicted in SEQ. ID. No: 3, ii) said first and second conserved primer of HLA-DQA1 are depicted in SEQ. ID. NO: 3 and SEQ. ID. NO: 7, and iii) said third conserved primer of HLA-DQ1 is depicted in SEQ. ID. NO: 14 or SEQ. ID. NO: 15, (g) wherein: i) said conserved oligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1, ii) said first and second conserved primers of HLA-DQB1 are depicted in SEQ. ID. NO: 1 and SEQ. ID. NO: 4, and iii) said third conserved primer of HLA-DBQ1 is depicted in SEQ. ID. NO. 10 or SEQ. ID. NO: 12; (h) wherein: i) said conserved oligonucleotide antisense primer of HLA-DPB1 is depicted in SEQ. ID. NO: 24, ii) said first and second conserved primers of HLA-DPB1 are depicted in SEQ. ID. NO: 24 and SEQ. ID. NO: 23, and iii) said third conserved primer of HLA-DPB1 is depicted in SEQ. ID. NO: 25 or SEQ. ID. NO: 26; and (i) wherein: i) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2, ii) said first and second conserved primers of HLA-DRB are depicted in SEQ. ID. NO: 2 and SEQ. ID. NO: 6, and iii) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13 or SEQ. ID. NO: 5.
4. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1; (b) said first conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1 and said first nonconserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 47 and wherein annealing of said conserved/nonconserved primer pair occurs at about 37.degree. C.; and (c) said third conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 4.
5. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1; (b) said first conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1 and said first nonconserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 46 and wherein annealing of said conserved/nonconserved primer pair occurs at about 55.degree. C.-60.degree. C.; and (c) said third conserved primer of [HLA-DQB!] HLA-DOB1 is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12.
6. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1; (b) said first conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1 and said first nonconserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 48; and (c) said third conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12.
7. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1; (b) said first conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 1 and said first nonconserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 49; and (c) said third conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 10 or SEQ. ID. NO: 12.
8. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DPB1 is depicted in SEQ. ID. NO: 25; (b) said first and second conserved primers of HLA-DPB1 are depicted in SEQ. ID. NO: 25 and SEQ. ID. NO: 25; and (c) said third conserved primer of HLA-DPB1 is depicted in SEQ. ID. NO: 26.
9. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DPA1 is depicted in SEQ. ID. NO: 31; (b) said first and second conserved primers of HLA-DPA1 are depicted in SEQ. ID. NO: 31 and SEQ. ID. NO: 32; and (c) said third conserved primer of HLA-DPA1 is depicted in SEQ. ID. NO: 33 or SEQ. ID. NO: 34.
10. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DPA1 is depicted in SEQ. ID. NO: 39; (b) said first and second conserved primers of HLA-DPA1 are depicted in SEQ. ID. NO: 39 and SEQ. ID. NO: 38; and (c) said third conserved primer of HLA-DPA1 is depicted in SEQ. ID. NO: 40 or SEQ. ID. NO: 41.
11. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DPA1 is depicted in SEQ. ID. NO: 33; (b) said first and second conserved primers of HLA-DPA 1 are depicted in SEQ. ID. NO: 33 and SEQ. ID. NO: 32; and (c) said third conserved primer of HLA-DPA1 is depicted in SEQ. ID. NO: 34.
12. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 42; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
13. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 43; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
14. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 44; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
15. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said amplifying has four discrete amplifying steps, and wherein in a first discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said second conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 6, and in a second discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO. 42, and in a third discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said second nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 43 and in a fourth discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said third nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 44; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13 or SEQ. ID. NO: 5.
16. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 45; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
17. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 2 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 46; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
18. The method according to claim 3 wherein: (a) said conserved oligonucleotide antisense primer of HLA-DRB is depicted in SEQ. ID. NO: 2; (b) said first and second conserved primers of HLA-DRB are depicted in SEQ. ID. NO: 2 and SEQ. ID. NO: 5; and (c) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 11 or SEQ. ID. NO: 13.
19. A method for determining the genotype of one or more HLA Class II polymorphic gene loci of interest selected from the group consisting of HLA-DRB1/3/4/5, --DQB1, --DQA1, DQA, --DPB1 and DPA1 of a subject in a sample containing subject genomic DNA using a combination of conserved and nonconserved oligonucleotide primers, said method comprising: (a) amplifying said genomic DNA by at least one amplifying reaction to generate sufficient first and second double stranded amplifying reaction products for sequencing, using in a separate first amplifying reaction for each of said gene loci of interest encoded by the 3'-end of the alpha 1 domain exon, an oligonucleotide primer pair comprising a first conserved primer and either a second conserved primer or a first nonconserved primer for each of said gene loci of interest; (b) sequencing directly at least one strand of said amplifying reaction product for each of said gene loci of interest to produce at least one sequencing ladder for each of said gene loci of interest, using in each sequencing reaction a third conserved oligonucleotide primer for each of said gene loci of interest whereby a nucleotide sequence is obtained for each sequencing ladder; (c) comparing said nucleotide sequence obtained from each sequencing ladder to known sequences for each gene locus of interest; (d) comparing said nucleotide sequence of each sequencing ladder obtained with a conserved oligonucleotide primer pair to the nucleotide sequence of each allele of such gene locus amplified with a conserved/nonconserved oligonucleotide primer pair, whereby the genotype of one or more HLA Class II gene loci is obtained; (e) wherein: i) said first and second conserved primers of HLA-DQB1 are depicted in SEQ. ID. NO: 8 and SEQ. ID. NO: 9, and ii) said third conserved primer of HLA-DQB1 is depicted in SEQ. ID. NO: 12; (f) wherein: i) said first and second conserved primers of HLA-DPB1 are depicted in SEQ. ID. NO: 27 and SEQ. ID. NO: 28, and ii) said third conserved primer of HLA-DPB1 is depicted in SEQ. ID. NO: 29 or SEQ. ID. NO: 30; (g) wherein: i) said first and second conserved primers of HLA-DPA1 are depicted in SEQ. ID NO: 35 and SEQ. ID. NO: 36, and ii) said third conserved primer of HLA-DPA1 is depicted in SEQ. ID. NO: 37; and (h) wherein: i) said first and second conserved primers of HLA-DRB are depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 5, and ii) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: or SEQ. ID. NO: 16.
20. The method according to claim 19 wherein: (a) said first and second conserved primers of HLA-DPB1 are depicted in SEQ. ID. NO: 27 and SEQ. ID. NO: 29; and (b) said third conserved primer of HLA-DPB1 is depicted in SEQ. ID. NO: 30.
21. The method according to claim 19 wherein: (a) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 43; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 13 and SEQ. ID. NO: 16.
22. The method according to claim 19 wherein: (a) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 44; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16.
23. The method according to claim 19 wherein: (a) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 42; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16.
24. The method according to claim 19 wherein: (a) said amplifying has four discrete amplifying steps and wherein in a first discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said second conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 5, and in a second discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 43, and in a third discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 44 and in a fourth discrete amplifying step said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said third nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 42; and b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 13 or SEQ. ID. NO: 16.
25. The method according to claim 19 wherein: (a) said first and second conserved primers of HLA-DRB are depicted in SEQ. ID. NO: 13 and SEQ. ID. NO: 22; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 22.
26. The method according to claim 19 wherein: (a) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 17 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 18; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 19.
27. The method according to claim 19 wherein: (a) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 45; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 21 or SEQ. ID. NO: 13.
28. The method according to claim 19 wherein: (a) said first conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 20 and said first nonconserved primer of HLA-DRB is depicted in SEQ. ID. NO: 46; and (b) said third conserved primer of HLA-DRB is depicted in SEQ. ID. NO: 21 or SEQ. ID. NO. 13.
29. A method according to claim 3 wherein: (a) said synthesizing step and said amplifying step for all of said gene loci of interest are performed using conserved oligonucleotide primers; and (b) said synthesizing step and said amplifying step for all of said gene loci of interest are performed in the same reaction tube.
30. A method according to claim 19 wherein: (a) said amplifying step for all of said gene loci of interest is performed using conserved oligonucleotide primers; and (b) said amplifying step for all of said gene loci of interest is performed in the same reaction tube.
31. A method according to claim 3-30 wherein: (a) each of said synthesizing steps for all of said gene loci of interest are performed simultaneously; (b) each of said amplifying steps for all of said gene loci of interest are performed simultaneously; and (c) each of said sequencing steps for all of said gene loci of interest are performed simultaneously.
32. A method according to claim 3-30 for rapid automated determination of HLA Class II genotype of said gene loci of interest of a subject in a sample containing subject nucleic acid, wherein: (a) said isolating step is performed with an RNA/DNA extractor; (b) said amplifying step is performed by at least one amplifying reaction using a thermocycler; (c) said sequencing step is performed in an automated sequencing apparatus with a sequencing enzyme and a conserved primer specific for each locus to be sequenced; and (d) said comparing is performed with a computer having a data base with allelic sequence information.
33. A method according to claim 3-30 for rapid automated determination of HLA Class II genotype of said gene loci of interest of a subject in a sample containing subject nucleic acid, wherein: (a) said isolating step is performed with an RNA/DNA extractor wherein all isolating steps for all gene loci of interest are performed simultaneously; (b) said amplifying step is performed by at least one amplifying reaction using a thermocycler wherein all amplifying steps for all gene loci of interest are performed simultaneously; and (c) said sequencing step is performed in an automated sequencing apparatus with a sequencing enzyme wherein all sequencing steps for all gene loci of interest are performed simultaneously; and (d) said comparing is performed with a computer having a data base with allelic sequence information.
34. The method of claim 3 or 19 wherein said comparing is conducted with a computer having a program including nucleotide sequences of all alleles of all known haplotypes for all of said loci to be genotyped.
35. The method of claim 3 or 19 where said amplifying with said conserved oligonucleotide primer includes annealing said conserved oligonucleotide primer of said isolated nucleic acid at about 37.degree. C.
36. The method of claim 3 wherein said amplifying with said non-conserved primer includes annealing said non-conserved primer to said cDNA at about 55.degree. C.
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