Method of Genotyping Blood Cell Antigens and Kit Suitable for Genotyping Blood Cell Antigens

Abstract

A method of genotyping blood cell antigens comprising subjecting DNA from an individual of a mammalian species to a multiplex Polymerase Chain Reaction (PCR) to amplify and detectably label a region of the locus of at least two different blood cell antigens which contains the site of nucleotide polymorphism of said blood cell antigen and using the thus amplified and labeled DNA fragments to determine the genotype for each of said blood cell antigens. The multiplex PCR -comprises the use of at least one pair of blood cell antigen-specific chimeric primers for each blood cell antigen to be genotyped and at least one detectably labeled universal primer, preferably a pair of detectably labeled universal primers. The universal primer(s) have a unique sequence not occurring in the DNA of said mammalian species. Each chimeric primer pair comprises a left chimeric primer and a right chimeric primer, Each of them comprising a blood cell antigen-specific part at the 3′ end and a universal part at the 5′ end. The base sequence of the universal part of the chimeric primers corresponds to the base sequence of said at least one universal primer. The blood cell antigen-specific parts of the chimeric primer pair enclose a region of the locus of the blood cell antigen which contains the site of nucleotide polymorphism of said blood cell antigen. A kit for genotyping blood cell antigens by this method. A set of blood cell antigen-specific chimeric primer pairs and a set of blood cell antigen allele-specific oligonucleotide probes.

Description

FIELD OF THE INVENTION

This invention is in the field of genotyping of blood cell antigens, more particularly antigens on red blood cells (blood group antigens), blood platelets (platelet antigens) and leukocytes (leukocyte antigens).

The present invention provides a method of genotyping blood cell antigens and a kit suitable for genotyping blood cell antigens, and provides sets of primers and probes useful for genotyping blood cell antigens.

BACKGROUND OF THE INVENTION

When blood, or a blood fraction, such as red blood cells (erythrocytes or red cells), platelets (thrombocytes) and white blood cells (leukocytes), derived from a donor are administered to another person (or more generally to another mammalian individual), serious adverse reactions may occur when the donor blood or blood fraction does not match properly with the blood of the recipient. Well known are transfusion reactions (agglutination) occurring when for example blood from a donor of blood group A is given to a person of blood group B (blood group antigens A and B belong to the AB0 system). When blood of a rhesus D (RhD) positive donor is given to a RhD negative patient there is a high chance that alloantibody formation occurs. RhD antibodies will lead to rapid destruction of RhD-positive red cells and to transfusion reactions. Furthermore, when a woman with red cell or platelet antibodies becomes pregnant, those antibodies can cross the placenta and can destruct the red cells or the platelets of the unborn child. This can lead to severe hemolysis resulting in anaemia, jaundice (after birth) and if not treated it can be fatal or lead to cerebral damage. Thus, to avoid transfusion reactions, the current blood transfusion policy is to transfuse only AB0 and RhD matched red cells. To avoid alloantibody formation with possible complications during pregnancy for women in childbearing age only AB0, RhD and K1 matched red cells are transfused. Possibly, in the future, also Rhc and RhE matched red cells will be given to women in childbearing age.

Various other blood group antigens (red cell antigens) exist, however, and these may also cause serious problems when non-matching donor blood is given to a recipient with alloantibodies. Typing of platelet-specific antigens is important for the diagnosis and therapy of the patients since different alloimmune thrombocytopenic syndromes can occur. If a woman has developed anti-platelet antigen antibodies (in most cases Human Platelet Antigen (HPA) type 1a antibodies) and mostly developed during a pregnancy, these antibodies can lead to fetal platelet destruction with an increased bleeding tendency in the unborn. In a number of cases this will lead to intracranial bleeding. To prevent bleeding, HPA-1a-negative platelets need to be transfused.

Platelets are usually transfused without previous typing of platelet antigens [human platelet antigens (HPA) in humans] of the donor and the recipient. Transfusions of non-matching blood or blood fractions may cause the generation of alloantibodies, especially in patients who need frequent or recurrent red cell or platelet (or leukocyte) transfusions. If multiple alloantibodies have formed, or if the alloantibodies are directed against high-frequency antigens, it can be a problem to find compatible red cells or platelets. According to a published study (Seltsam et al., 2003), transfusion support was unsatisfactory in about one-third of the hospitalized patients with antibodies to high-frequency antigens.

The classical method of testing for blood group antigens and antibodies is phenotyping by the hemagglutination test. The technique of this serological test is simple and inexpensive, but its costs and difficulties increase when multiple assays need to be done for complete typing and it requires availability of a large number of specific antisera. In The Netherlands alone, the total number of blood donors is in the order of magnitude of 500,000 people and it is estimated that the number of donors increases each year with about 60,000 new donors. For red cells, there are at least 29 blood group antigen systems (each with a number of different alleles). Furthermore, it will be relevant to type for high-frequency antigens and low-frequency antigens. The number of clinically relevant blood cell antigen systems is about 60. Complete phenotyping of all blood donors is therefore expensive, laborious, time consuming and not feasible due to lack of sufficient typing reagents.

The molecular basis for most blood cell antigen systems is known. Most blood group antigens, platelet antigens and neutrophil antigens are bi-allelic and are the result of a single nucleotide polymorphism (SNP). These SNPs may be used for genotyping. Innumerable DNA-based assays have been described in the scientific literature for the genotyping of blood groups and platelet antigens. These include PCR-RFLP, allele-specific PCR, sequence specific PCR as single or multiplex assays, real-time quantitative PCR, a single-nucleotide dye terminator extension method and high-throughput bead technology (reviewed by Reid, 2003). Semiautomated methods using a mass spectrophotometer or pyrosequencer may also be used for genotyping.

For example, Randen et al. (2003) recently disclosed a genotyping of the human platelet antigens 1, 2, 3, 4, 5 and Gov (recently called HPA-15, Metcalfe et al., 2003) by melting curve analysis using LightCycler technology. The biallelic systems HPA-1 through 5 and Gov are the ones most frequently involved in disease (Berry et al., 2000), making them important targets for genotyping. The LightCycler technology involves an amplification of relevant fragments of donor DNA by PCR using a specific primer pair for each of the above mentioned platelet antigens. By using fluorescent hybridization probes and melting curve analysis it was possible to achieve a simultaneous detection of both alleles of a platelet antigen in the same capillary, without a need of the laborious and time consuming gel electrophoresis of earlier genotyping methodology.

However, as with all other described blood cell antigen genotyping methods, also the LightCycler technology is not capable of performing the enormous task of a complete genotyping of blood donors, which would require methodology which is suitable for high-throughput screening. Typing each year 60,000 donors after two different donations for 60 blood group and platelet antigens would involve more than 7 million typing tests per year or about 140,000 typing tests per week. A suitable high-throughput method, which is rapid and reliable, is highly desirable for this task.

Multiplex Polymerase Chain Reaction

The technique of Polymerase Chain Reaction (PCR) has developed much since it was introduced in the 1980's. Chamberlain et al. (1988) taught multiplex PCR as a general technique for the amplification of multiple loci in (genomic) DNA. Herein, instead of one primer pair for the amplification of one locus, more primer pairs are added in one reaction mixture. However, the development of such multiplex PCRs is limited by the complexity of the amplification reaction. Reaction components and cycling conditions must be adjusted for each extra pair of primers. Shuber et al. (1995) introduced ‘chimeric’ sequence-specific primers to circumvent this problem. These primers are complementary to the template DNA and contain an unrelated 20-nucleotide tag at the 5′ end (universal sequence). Although the primers were designed in such a way that their predicted melting temperatures are similar to those of the other primers and have a calculated AG for primer duplexing below −10 kcal/mole, the concentration of the primers still needs to be adjusted to obtain similar yields of PCR product. To circumvent this problem and to also reduce primer-dimer formation further, Belgrader et al. (1996) applied only a limited amount of chimeric primer (2 pmol) and added an overload of universal primer after 15 PCR cycles. Thermal cycling proceeded then for another 25 cycles at a lower annealing temperature. Brownie et al. (1997) included the universal primer already in the PCR reaction mixture at the start. After 4 cycles of PCR the annealing temperature is raised from 60° C. to 74° C. and another 35 cycles of PCR is performed. Further, a nested PCR is taught by Heath et al. (2000) who applies in the second PCR two (complementary) universal primers, one of them carrying a fluorescent tag attached to the 5′ end.

So far, multiplex PCR has only been used with relatively small sets of primer pairs for allele-specific amplification. Neither the feasibility of using a multiplex PCR in a method of genotyping a large number of blood cell antigens, nor the nature of the PCR conditions and the primer mixtures that are suitable for such blood cell antigen genotyping, nor a practical, rapid and reliable method for analyzing the products of the amplification to assign the clinically relevant blood cell antigen genotypes have been described in the prior art.

DNA Microarrays

Several approaches using microarray technology are known in the general field of genotyping. One of these approaches is the so-called mini-sequencing method, which includes an allele-specific extension either on the microarray or in solution (Pastinen et al., 1997; Fan et al., 2000). A difficulty in this approach is the occurrence of non-specific primer extension and further optimization of primers or the extension method itself is necessary (Pastinen et al., 2000; Lindroos et al., 2002). Moreover, this method requires laborious steps of enzymatic treatment and purification.

Another approach is called allele-specific oligonucleotide hybridization (ASO) on microarrays. This approach relies on the thermal stability of the target and short oligonucleotide probes for genotype determination (Hacia et al., 1996; Wang et al., 1998). The first light-generated oligonucleotide arrays were developed in 1991 (Fodor et al.). The method has been used for the determination of new SNPs or for resequencing. It has also been used for genotyping (Evans et al., 2002) by spotting the PCR amplification products derived from patient samples into an array and contacting the array with allele-specific oligos to discriminate the alleles of the patient. Many variations have been described, using different kinds of tools, like enzymes, nanoparticle probes, artificial nucleotides, thermal gradients, flow-through arrays, blocking oligonucleotides, for improving the sensitivity or specificity (Lu et al., 2002; Park et al., 2002; Prix et al., 2002; Kajiyama et al., 2003; Jobs et al., 2003; Van Beuningen et al., 2001; Iwasaki et al., 2002). Wen et al., 2000, made a comparison of the sensitivity and specificity of an oligonucleotide array analysis of TP53 mutations with conventional DNA sequence analysis. The oligonucleotide array used contained a plurality of probes per SNP. To immobilize the oligos to the matrix support, usually a glass slide, the probes may be provided with a spacer and an amine group (Guo et al., 2001; Wen et al., 2003). Various microarray formats have been described, for example slides with a 96-microarray format containing 250 spots per array to improve throughput (Huang et al., 2001). Flow-through systems have been described in order to reduce the hybridization time and thereby increase throughput (Cheek et al., 2001; Van Beuningen et al., 2001).

So far, DNA microarray methods have not been applied to genotyping of blood cell antigens and neither the feasibility of using a DNA microarray in a method of genotyping a large number of blood cell antigens, nor the nature of the oligonucleotide probes and microarray formats that are suitable for such blood cell antigen genotyping, nor a practical, rapid and reliable method for analyzing the hybridization results to assign the clinically relevant blood cell antigen genotypes have been described in the prior art.

SUMMARY OF THE INVENTION

An object of the present invention is to provide methods and means allowing a practical, rapid and reliable genotyping of a large number of blood cell antigens.

Another object of the invention is to develop a high-throughput technique which allows genotyping of the whole existing donor cohort and/or the donor cohort increase for a number of blood cell antigens in the order of magnitude of 20 and ultimately even some 60 blood cell antigen systems, to thereby facilitate the selection of correct donor blood and improve the safety of blood transfusion.

Again another object of the invention is to achieve an essentially complete and reliable genotyping, at least covering the majority of clinically relevant blood cell antigen systems, using simple apparatus, in a short time, such as less than 30 hours, or less than 24 hours, preferably less than 6 hours and more preferably less than 2 hours or even less than 1 hour, on a single DNA sample subjected to one PCR reaction in a single reaction tube to simultaneously amplify and detectably label relevant DNA fragments.

These objects are achieved by the present invention which provides, in one aspect, a method of genotyping blood cell antigens comprising subjecting DNA from an individual of a mammalian species to a multiplex Polymerase Chain Reaction (PCR) to amplify and detectably label a region of the locus of at least two different blood cell antigens which contains the site of nucleotide polymorphism of said blood cell antigen and using the thus amplified and labeled DNA fragments to determine the genotype for each of said blood cell antigens, said multiplex PCR comprising the use of at least one pair of blood cell antigen-specific chimeric primers for each blood cell antigen to be genotyped and at least one detectably labeled universal primer, wherein said at least one universal primer has a unique sequence not occurring in the DNA of said mammalian species, and wherein each chimeric primer pair comprises a left chimeric primer and a right chimeric primer, each of them comprising a blood cell antigen-specific part at the 3′ end and a universal part at the 5′ end, wherein the base sequence of the universal part of the chimeric primers corresponds to the base sequence of said at least one universal primer, and wherein said blood cell antigen-specific parts of the chimeric primer pair enclose a region of the locus of the blood cell antigen which contains the site of nucleotide polymorphism of said blood cell antigen.

In the method of the invention, it is preferred that a pair of detectably labeled universal primers with a unique sequence not occurring in the DNA of said mammalian species is used and that for each chimeric primer pair the base sequence of the universal part of one member of the chimeric primer pair corresponds to the base sequence of one member of the universal primer pair and the base sequence of the universal part of the other member of the chimeric primer pair corresponds to the base sequence of the other member of the universal primer pair.

Further, it is strongly preferred in the present invention that the genotype for each of said blood cell antigens is determined by hybridizing the products of the multiplex PCR amplification, after denaturation, to blood cell antigen allele-specific oligonucleotide probes contained in a DNA array or presented in any other form, such as supported on beads, and analyzing the hybridization pattern.

In another aspect, the present invention provides a kit for genotyping blood cell antigens by a method as defined herein, comprising at least one pair of blood cell antigen-specific chimeric primers for each blood cell antigen to be genotyped and one pair of detectably labeled universal primers, both as defined herein.

This invention further provides a set of blood cell antigen-specific chimeric primer pairs useful in a multiplex PCR, and a set of blood cell antigen allele-specific oligonucleotide probes useful for genotyping blood cell antigens.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the results of a multiplex PCR of 19 blood group antigens with or without the presence of universal MAPH primers. Very little amount of PCR product is amplified during the first amplification cycles (lane 2), but enough to be used as template by the universal MAPH primers in the following cycles. The MAPH primers perform the actual amplification (lane 3), so adjustments of primer concentrations are hardly necessary and similar yields of PCR product are obtained.

FIG. 2 depicts the pattern obtained with ABI capillary sequencer from the multiplex PCR of 19 blood group antigens as described in this invention.

FIG. 3 depicts the scan results of the hybridisation of 6 donor samples, each comprising the 6 human platelet antigen 1 through 5 and Gov PCR products. The PCR products of one sample were hybridised to two arrays, that is 4 blocks. The original scans have red spots and a black background. For better visualisation this is changed to grayscale and inverted.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method of genotyping blood cell antigens comprising subjecting DNA from an individual of a mammalian species to a multiplex Polymerase Chain Reaction (PCR) to amplify and detectably label a region of the locus of at least two different blood cell antigens which contains the site of nucleotide polymorphism of said blood cell antigen and using the thus amplified and labeled DNA fragments to determine the genotype for each of said blood cell antigens, said multiplex PCR comprising the use of at least one pair of blood cell antigen-specific chimeric primers for each blood cell antigen to be genotyped and at least one detectably labeled universal primer, wherein said at least one universal primer has a unique sequence not occurring in the DNA of said mammalian species, and wherein each chimeric primer pair comprises a left chimeric primer and a right chimeric primer, each of them comprising a blood cell antigen-specific part at the 3′ end and a universal part at the 5′ end, wherein the base sequence of the universal part of the chimeric primers corresponds to the base sequence of said at least one universal primer, and wherein said blood cell antigen-specific parts of the chimeric primer pair enclose a region of the locus of the blood cell antigen which contains the site of nucleotide polymorphism of said blood cell antigen.

More particularly, the invention provides a method of genotyping blood cell antigens comprising subjecting DNA from an individual of a mammalian species to a multiplex Polymerase Chain Reaction (PCR) to amplify and detectably label a region of the locus of at least two different blood cell antigens which contains the site of nucleotide polymorphism of said blood cell antigen and using the thus amplified and labeled DNA fragments to determine the genotype for each of said blood cell antigens, said multiplex PCR comprising the use of at least one pair of blood cell antigen-specific chimeric primers for each blood cell antigen to be genotyped and one pair of detectably labeled universal primers, wherein said pair of universal primers comprises a forward universal primer and a reverse universal primer each having a unique sequence not occurring in the DNA of said mammalian species, and wherein each chimeric primer pair comprises a left chimeric primer and a right chimeric primer, each of them comprising a blood cell antigen-specific part at the 3′ end and a universal part at the 5′ end, wherein the base sequence of the universal part of one of these chimeric primers corresponds to the base sequence of one of the universal primers and the base sequence of the universal part of the other chimeric primer corresponds to the base sequence of the other of the universal primers, and wherein said blood cell antigen-specific parts of the chimeric primer pair enclose a region of the locus of the blood cell antigen which contains the site of nucleotide polymorphism of said blood cell antigen.

The invention involves a particular sequence of steps that in combination allows to achieve the above mentioned objects. This sequence of steps comprises isolation of DNA from a sample which is to be subjected to the genotyping analysis of this invention, followed by one of the key aspects of the present invention, a multiplex PCR of gene fragments carrying the single nucleotide polymorphisms (SNPs) that are responsible for blood cell antigen differences. This multiplex PCR applies a mixture of different primers, including two or more pairs of chimeric primers and in addition including a labelled universal primer or a pair of labelled universal primers. Then the thus amplified and labelled DNA fragments are denatured (such as by heating) and are contacted with a set of probes, preferably contained in a microarray comprising a multitude of allele-specific probes, under conditions which support hybridisation between matching sequences. In the next step, the presence of labelled fragments is measured for each spot in the microarray, and finally the signals measured are interpreted to type the sample. The probes may be presented also in other formats, for example as probes supported on beads (glass beads, polystyrene beads, etc.), or probes attached to the wall of a suitable container, etc. An array format is however the most preferred embodiment.

Species

The invention allows the genotyping of blood cell antigens not only of humans, but of all animals with a blood cell antigen system. For all practical purposes, genotyping will relate to blood cell antigens of mamnmals, including for example farm animals and pets, wild-life animals and generally all animals of economic, emotional or wild life preservation value. Most preferably, however, the invention concerns the genotyping of human blood cell antigens.

Blood Cell Antigens

The blood cell antigens that may be genotyped by the present invention include typical blood group antigens, such as Kidd, Duffy, Kell and Rhesus. The blood group antigens of the AB0 system may be genotyped by the present invention as well, although it may be necessary, because of their extreme importance, to perform the existing and well-accepted serological test. Other blood group antigens that may be genotyped by the present invention comprise antigen variants and or gene variants leading to gene silencing of the blood group systems MNSs, Rhesus (Rh), Kell, Kidd, Duffy, Colton, Diego, Dombrock, Lutheran, Lewis, Cartwright, Landsteiner Wiener (LW), Cromer, Knops, Kx, Indian, Gerbich, Hh, Chido/Rodgers, GIL, I, JMH, OK, P-related, RAPH-MER, Scianna, Xg, YT, etc.

In addition to the blood group antigens, also platelet and leukocyte (especially neutrophil) antigens may be genotyped by the method of this invention. The clinically most relevant platelet antigens, at least in the Western world, comprise HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and Gov (or HPA-15). Other platelet antigens that may be genotyped by the present invention, even though they are considered of lesser importance, comprise for example human platelet antigens 6 through 14 and 16. Examples of typical Human Neutrophil Antigens that may be genotyped by the method of the invention include HNA-1, 4 and 5. Others, such as HNA-2 and 3, can be genotyped as soon as their molecular basis has been elucidated.

It is conceived that the present invention is useful even when only a restricted number of blood cell antigens is genotyped. It may for some purposes be sufficient that only the red cell antigens are genotyped, or only the platelet antigens, or only a selected group of antigens which is relevant in a certain context. The minimum number of blood cell antigens to be genotyped by the present invention is two blood cell antigens, for example the blood cell antigen RhD and the RHD gene variant RhDΨ, or the blood cell antigen JK1/2 (Jk^a/Jk^b) and the blood cell antigen FY1/2 (Fy^a/Fy^b). Genotyping of a larger number of blood cell antigens is clearly preferred, for example three, four, five, and especially six or more. For most purposes, however, a more or less complete genotyping, at least covering most of the clinically relevant blood cell antigens, is preferred. Most preferred, the invention includes at least six different chimeric primer pairs specific for HPA's 1 through 5 and HPA-15, and at least 10 or 11 or 12 chimeric primer pairs specific for red cell antigens, such as the various Kidd, Duffy, Kell and Rhesus antigens.

The DNA

The DNA subjected to the multiplex PCR will usually be genomic DNA of the individual whose blood cell antigens are to be genotyped The donor DNA may be obtained from any suitable source, for example from EDTA, heparin or citrate treated blood samples or buffy coats, using isolation procedures and means well known to the skilled person, such as for example the commercially available Qiagen Blood DNA extraction kit, use of a salting-out method, such as by the standard protocol of Miller et al. (1988), or use of the Roche Magnapure.

Universal Primers

The method of the invention uses at least one universal primer carrying a detectable label, preferably a pair of universal primers both carrying a detectable label. The sequence of the universal primer(s) corresponds with the sequence of the universal part at the 5′ end of the chimeric primers. It is possible to use only one universal primer. In that case, all chimeric primers have an identical base sequence at their 5′ end, i.e. the universal part at the 5′ end of the chimeric primers corresponds with the sequence of said single universal primer. Practically, however, it was found preferable to use two different universal primers (i.e. a pair of universal primers). In that case, each pair of chimeric primers contains a primer with a universal part corresponding to one of the universal primers and a primer with a universal part corresponding to the other universal primer.

It is important that the universal primer(s) do not hybridize to the DNA which is to be genotyped. Assuming that the DNA to be genotyped is human genomic DNA, the universal primer(s) (and the corresponding parts of the chimeric primers) should not hybridize with human genomic DNA and therefore have a unique sequence not occurring in said DNA. Preferably, the sequence of the universal primer(s) differs significantly from any sequence occurring in the human genome. Further, it is strongly preferred that the universal primer(s) are designed such that there Tm value is similar to the Tm value of the blood cell antigen specific part of the chimeric primers. Preferably, the Tm value of the universal primer(s) is between 50 and 70° C., more preferably between 56 and 68° C. It is also preferred that the universal primer(s) have a length of between 12 and 30 bases, more preferably between 15 and 25 or even between 16 and 20 bases.

Very good results were obtained with universal primers disclosed in White et al., 2002, with the sequences ggccgcgggaattcgatt (SEQ ID NO:1, forward MAPH), with a Tm of 67.55° C., and gccgcgaattcactagtg (SEQ ID NO:2, reverse MAPH) with a Tm of 57.94° C.

Detectable Labels

The universal primer(s) carry a detectable label. The label may be any label suitable for the purpose, such as a radioactive atom or group, an enzyme, such as an enzyme catalyzing a measurable conversion of a substrate thereof, a dye, a fluorescent substance, a chemiluminescent substance, biotin, etc. Most preferably, the label is a fluorescent group, of which many are known to the person skilled in the art. Examples thereof are Cy3, Cy5, Fluorescein (FITC), Phycoerythrin (PE), Rhodamine, Hex, Tet, Fam, etc.

Most preferably, the invention uses the fluorescent group Cy5, which is a sulfoindocyanine dye (Mujumdar et al., 1993).

Usually, in particular when the label is a moiety attached to the oligonucleotide, it will be attached to the 5′ end of the oligonucleotide sequence of the universal primer(s).

Because the universal primer(s) are labeled, while the chimeric primers are not labelled, the invention achieves very efficient labeling simultaneous with amplification, while the various chimeric primers can be used without previous labeling.

Chimeric Primers

The chimeric primers used in this invention have a universal part at their 5′ end and a blood cell antigen-specific part at their 3′ end. The oligonucleotide sequence of the universal part corresponds with the sequence of the universal primer(s) and requires no further elucidation here.

As to the blood cell antigen-specific part of the chimeric primers, it is most preferable that these parts have similar Tm values, both in comparison with blood cell antigen-specific parts of other chimeric primers in the mixture, and in comparison with the universal primers used. Thus, it is preferred that these blood cell antigen-specific parts of the chimeric primers have Tm values between 50 and 70° C., more preferably between 55 and 65° C. and most preferably between 56 and 62° C. It is also preferred that the blood cell antigen-specific parts of the chimeric primers have a length of betveen 12 and 35 bases, more preferably between 15 and 30 or even between 16 and 25 bases.

It is further preferred that the blood cell antigen-specific parts of the chimeric primers are selected in such a way that the PCR amplification results in oligonucleotide products with a length of between 50 and 800, preferably between 80 and 500, most preferably between 100 and 400 or 300 nucleotides. A product length of from 100 to 200 nucleotides would be ideal.

Various software products are available that can be used to design suitable blood cell antigen-specific parts of the chimeric primers. On the basis of the publicly available DNA sequences of the blood cell antigen-encoding genes, the software product Primer3 (http://www.broad.mit.edu/cgi-bin/primer/primer3 www.cgi) allows to design primers. To check for any unspecific genomic DNA binding the CELERA database can be used and to check for any primer-dimer formation the Oligo6 software (Medprobe) can be used. It is preferred to select primers having a calculated ΔG below −10 kcal/mole.

A list of preferred chimeric primers includes the following:

HPA1-left	gccgcgaattcactagtgcttcaggtcacagcgaggt	SEQ ID NO:3
HPA1-right	ggccgcgggaattcgattgctccaatgtacggggtaaa	SEQ ID NO:4
HPA2-left	gccgcgaattcactagtgtgaaaggcaatgagctgaag	SEQ ID NO:5
HPA2-right	ggccgcgggaattcgattagccagactgagcttctcca	SEQ ID NO:6
HPA3-left	gccgcgaattcactagtggcctgaccactcctttgc	SEQ ID NO:7
HPA3-right	ggccgcgggaattcgattggaagatctgtctgcgatcc	SEQ ID NO:8
HPA4-left	gccgcgaattcactagtgatccgcaggttactggtgag	SEQ ID NO:9
HPA4-right	ggccgcgggaattcgattccatgaaggatgatctgtgg	SEQ ID NO:10
HPA5-left	gccgcgaattcactagtgtccaaatgcaagttaaattaccag	SEQ ID NO:11
HPA5-right	ggccgcgggaattcgattacagacgtgctcttggtaggt	SEQ ID NO:12
HPA15-left	gccgcgaattcactagtgtgtatcagttcttggttttgtgatg	SEQ ID NO:13
HPA15-right	ggccgcgggaattcgattaaaaccagtagccacccaag	SEQ ID NO:14
JK1/2-left	gccgcgaattcactagtggtctttcagccccatttgag	SEQ ID NO:15
JK1/2-right	ggccgcgggaattcgattgttgaaaccccagagtccaa	SEQ ID NO:16
FY1/2-left	gccgcgaattcactagtggaattcttcctatggtgtgaatga	SEQ ID NO:17
FY1/2-right	ggccgcgggaattcgattaagaagggcagtgcagagtc	SEQ ID NO:18
GATAbox-left	gccgcgaattcactagtgggccctcattagtccttgg	SEQ ID NO:19
GATAbox-right	ggccgcgggaattcgattgaaatgaggggcatagggata	SEQ ID NO:20
Fyx-left	gccgcgaattcactagtgtcatgcttttcagacctctcttc	SEQ ID NO:175
Fyx-right	ggccgcgggaattcgattcaagacgggcaccacaat	SEQ ID NO:176
KEL1/2-left	gccgcgaattcactagtgaagggaaatggccatactga	SEQ ID NO:21
KEL1/2-right	ggccgcgggaattcgattagctgtgtaagagccgatcc	SEQ ID NO:22
KEL3/4-left	gccgcgaattcactagtggcctcagaaactggaacagc	SEQ ID NO:23
KEL3/4-right	ggccgcgggaattcgattagcaaggtgcaagaacactct	SEQ ID NO:24
KLBL6/7-left	gccgcgaattcactagtggcagcaccaaccctatgttc	SEQ ID NO:177
KEL6/7-right	ggccgcgggaattcgatttcaggcacaggtgagcttc	SEQ ID NO:178
RHCEex2for	gccgcgaattcactagtgcgtctgcttccccctcc	SEQ ID NO:25
RHex2rev	ggccgcgggaattcgattctgaacagtgtgatgaccacc	SEQ ID NO:26
RHDex3-left	gccgcgaattcactagtgtcctggctctccctctct	SEQ ID NO:179
RHCEex3-right	ggccgcgggaattcgatttttttcaaaaccccggaag	SEQ ID NO:180
RHCEex5-left	gccgcgaattcactagtgggatgttctggccaagtg	SEQ ID NO:27
RHex5rev	ggccgcgggaattcgattggctgtcaccacactgactg	SEQ ID NO:28
RHDΨ-left	ggccgcgggaattcgattgtagtgagctggcccatca	SEQ ID NO:29
RHDΨ-right	gccgcgaattcactagtgtgtctagtttcttaccggcaagt	SEQ ID NO:30
RHD-leftB	gccgcgaattcactagtgttataataacacttgtccacaggg	SEQ ID NO:31
RHD-rightC	ggccgcgggaattcgattcggctccgacggtatc	SEQ ID NO:32
BigC-left	gccgcgaattcactagtgggccaccaccatttgaa	SEQ ID NO:33
BigC-rightintron2	ggccgcgggaattcgattccatgaacatgccacttcac	SEQ ID NO:34
RhDVI-left (fw)	ggccgcgggaattcgattctttgaattaagcacttcacaga	SEQ ID NO:181
RhDVI-right (rev)	gccgcgaattcactagtggccagaatcacactcctgct	SEQ ID NO:182
MN-left	gccgcgaattcactagtgtgagggaatttgtcttttgca	SEQ ID NO:35
MN-right	ggccgcgggaattcgattcagaggcaagaattcctcca	SEQ ID NO:36
Ss-left	gccgcgaattcactagtgtttttctttgcacatgtcttt	SEQ ID NO:183
Ss-right	ggccgcgggaattcgatttctttgtctttacaatttcgtgtg	SEQ ID NO:184
U-left	gccgcgaattcactagtgcgctgatgttatctgtcttatttttc	SEQ ID NO:37
U-right	ggccgcgggaattcgattgatcgttccaataataccagcc	SEQ ID NO:38
DO-left	ggccgcgggaattcgatttgatccctccctatgagctg	SEQ ID NO:185
DO-right	gccgcgaattcactagtgttatatgtgctcaggttcccagt	SEQ ID NO:186
JO-left	gccgcgaattcactagtgcctggcttaaccaaggaaaa	SEQ ID NO:39
JO-right	ggccgcgggaattcgatttcatactgctgtggagtcctg	SEQ ID NO:40
Colton-left	gccgcgaattcactagtggccacgaccctctttgtct	SEQ ID NO:187
Colton-right	ggccgcgggaattcgatttacatgagggcacggaagat	SEQ ID NO:188
Diego-left	gccgcgaattcactagtgacttattcacgggcatccag	SEQ ID NO:189
Diego-right	ggccgcgggaattcgattaagctccacgttcctgaaga	SEQ ID NO:190
Wr-left	gccgcgaattcactagtgggcttcaaggtgtccaactc	SEQ ID NO:636
Wr-right	ggccgcgggaattcgattaggatgaagaccagcagagc	SEQ ID NO:637
Yt-left	ggccgcgggaattcgattccttcgtgcctgtggtagat	SEQ ID NO:638
Yt-right	gccgcgaattcactagtgttctgggacttctgggaatg	SEQ ID NO:639
Lu-left	gccgcgaattcactagtgggacccagagagagagagactg	SEQ ID NO:640
Lu-right	ggccgcgggaattcgattgggagtccagctggtatgg.	SEQ ID NO:641

Most preferred are the chimeric primers with SEQ ID NOS:3 to 40.

The above mentioned software products would allow the skilled person to design additional primers for other blood cell antigens. The present invention encompasses the use of a set of similar chimeric primers having the same blood cell antigen-specific part but a different universal part.

Proportions of the Primers

To achieve the aims of the invention, and in particular to avoid the occurrence of primer-dimers as much as possible, it is important that only a minimal proportion of the chimeric primers is used and that amplification is due to a large extent to elongation of the universal primers. Practically, it is preferred that the universal primers are used in a molar amount which is at least 10 times, more preferably at least 40 times the molar amount of each chimeric primer. Per amplification reaction 5 nM of each chimeric primer and 0.2 μM of each universal primer per chimeric primer pair is preferred. Per microliter reaction volume, an amount of 5 femtomol (5*10⁻¹⁵ mol) of each chimeric primer would be preferred, while about 0.2 pmol (0.2*10⁻¹² mol) of each universal primer is used per chimeric primer pair.

Multiplex PCR Conditions

The multiplex PCR in the method of this invention uses a mixture of universal and chimeric primers which are present from the beginning of the reaction. The PCR as used in this invention does not distinguish between a separate first part in which only chimeric primers are extended, and a second part in which only universal primers are extended. The present invention applies an annealing or primer extension temperature in the later cycles of the PCR which is the same as the annealing or primer extension temperature used in the first few cycles of the PCR. The inventors found that a change of reaction conditions in order to switch from chimeric primer extension to universal primer extension is not required.

The DNA polymerase used in the multiplex PCR is preferably a heat-resistant DNA polymerase, such as Taq DNA polymerase. Other heat-resistant DNA polymerases are useful as well, and even heat-sensitive DNA polymerases, although this may require repeated addition of a fresh amount of DNA polymerase. Suitable DNA polymerases are commercially available, for example as a component of multiplex PCR kits, like the Qiagen multiplex kit. Such kits also contain other necessary components, such as the required dNTP's and a suitable buffer system.

Apparatus for carrying out PCR thermocycling is commercially available as well, for example the MWG AG Biotech PrimusHT thermal cycler.

The amplification of the invention usually starts with a heat treatment to activate the DNA polymerase, for example 15 min at 95° C. Subsequently, the thermal cycling begins. Each cycle includes a heat denaturation step, an annealing step (to bind primer by hybridization to its template) and a primer extension or elongation step. In the invention, it is preferred that the heat denaturation step involves heating at a temperature of 90 to 98° C., more preferably at about 94 or 95° C., and takes a time of from 15 to 60 seconds (shorter and especially longer times are allowable), more preferably about 30 seconds. In the invention, it is further preferred that the annealing step involves heating at a temperature of 54 to 60° C., more preferably at about 57° C., and takes a time of from 60 to 120 seconds (shorter and especially longer times are allowable), more preferably about 90 seconds. In the invention, it is further preferred that the primer extension step involves heating at a temperature of 68 to 76° C., more preferably at about 72° C., and takes a time of from 60 to 120 seconds (shorter and especially longer times are allowable), more preferably about 90 seconds. A final treatment at the temperature of the primer extension step for a much longer time, for example 10 minutes, may conclude the thermal cycling.

The total number of cycles may be chosen at will, depending on the available time and the precision sought. A total of 30 cycles may be enough, but 40 or 45 or 50 cycles or more is preferable.

Genotyping

The result of the multiplex PCR is a mixture of labelled amplification products, in which ideally each product essentially comprises a region of the locus of different blood cell antigens which contains the site of nucleotide polymorphism of said blood cell antigen. In most cases of blood cell antigen polymorphisms that are presently known, the nucleotide polymorphism is a single nucleotide polymorphism (SNP), but polymorphism sites covering more than one nucleotide are encompassed as well.

To analyse the SNP's present in the mixture of labelled amplification products, various strategies are available, including sequencing of the products, but many of these are not suitable for rapid and reliable high-throughput genotyping. The present invention therefore preferably applies the special step of contacting the products of the multiplex PCR with a DNA array which contains blood cell antigen allele-specific oligonucleotide probes. Said contacting is done after denaturation, for example by heating, of the oligonucleotidic products of thie amplification and under conditions suitable for hybridization of these products to corresponding probes in the array. Purification of the products, of the amplification before contacting them with the probes is not necessary and will usually be omitted.

Probes

The probes are oligonucleotides including the site of the nucleotide polymorphism. Thereby, they are allele-specific. Although their length may vary from only 8 or 10 nucleotides up to several hundreds of nucleotides, it is preferred in this invention to use probes with a length of from 15 to 40 nucleotides, more preferably 16 or 17 to 29 or 30 nucleotides. Preferably, all probes have similar Tm values, in particular in a range of 55 to 75° C., more preferably in the range of 60 to 70° C. or especially in the range of 60 to 65° C.

Although in principle it is possible to use only one probe for each allele of each blood cell antigen, the invention applies several probes for each allele of each blood cell antigen, thereby increasing the reliability of the method. According to this invention, the array contains for each allele of each blood cell antigen at least two, preferably at least five, different sense probes and at least two, preferably at least five, antisense probes, each of them covering the site of the nucleotide polymorphism but at varying positions. Preferably, these positions are in or near the centre of the oligos.

Various software products, as mentioned above for the blood cell antigen-specific parts of the primers, are available that can be used to design suitable blood cell antigen allele-specific probes (in particular the sofware product Primer3).

A list of preferred probes includes the following (SNPs are highlighted):

HPA1-allele a:
HPA-1aa	tacaggccctgcctctggg	SEQ ID NO:41
HPA-1ab	aggccctgcctctgggct	SEQ ID NO:42
HPA-1ac	ccctgcctctgggctcacc	SEQ ID NO:43
HPA-1ad	tgcctctgggctcacctcg	SEQ ID NO:44
HPA-1ae	ctctgggctcacctcgctg	SEQ ID NO:45
HPA-1aa CR	cagcgaggtgagcccagag	SEQ ID NO:46
HPA-1ab CR	cgaggtgagcccagaggca	SEQ ID NO:47
HPA-1ac CR	ggtgagcccagaggcaggg	SEQ ID NO:48
HPA-1ad CR	agcccagaggcagggcct	SEQ ID NO:49
HPA-1ae CR	cccagaggcagggcctgta	SEQ ID NO:50
HPA1-allele b:
HPA-1ba	tacaggccctgcctccggg	SEQ ID NO:51
HPA-1bb	aggccctgcctccgggct	SEQ ID NO:52
HPA-1bc	ccctgcctccgggctcac	SEQ ID NO:53
HPA-1bd	ctgcctccgggctcacct	SEQ ID NO:54
HPA-1be	ctccgggctcacctcgct	SEQ ID NO:55
HPA-1ba CR	agcgaggtgagcccggag	SEQ ID NO:56
HPA-1bb CR	aggtgagcccggaggcag	SEQ ID NO:57
HPA-1bc CR	gtgagcccggaggcaggg	SEQ ID NO:58
HPA-1bd CR	agcccggaggcagggcct	SEQ ID NO:59
HPA-1be CR	cccggaggcagggcctgta	SEQ ID NO:60
HPA2-allele a:
HPA-2aa	ctgacgcccacacccaag	SEQ ID NO:61
HPA-2ab	ctcctgacgcccacaccc	SEQ ID NO:62
HPA-2ac	ggctcctgacgcccacac	SEQ ID NO:63
HPA-2ad	agggctcctgacgcccac	SEQ ID NO:64
HPA-2ae	ccagggctcctgacgccc	SEQ ID NO:65
HPA-2aa CR	cttgggtgtgggcgtcag	SEQ ID NO:66
HPA-2ab CR	gggtgtgggcgtcaggag	SEQ ID NO:67
HPA-2ac CR	gtgtgggcgtcaggagcc	SEQ ID NO:68
HPA-2ad CR	gtgggcgtcaggagccct	SEQ ID NO:69
HPA-2ae CR	gggcgtcaggagccctgg	SEQ ID NO:70
HPA2-allele b:
HPA-2ba	cctgatgcccacacccaag	SEQ ID NO:71
HPA-2bb	ctcctgatgcccacaccca	SEQ ID NO:72
HPA-2bc	ggctcctgatgcccacacc	SEQ ID NO:73
HPA-2bd	agggctcctgatgcccaca	SEQ ID NO:74
HPA-2be	ccagggctcctgatgccc	SEQ ID NO:75
HPA-2ba CR	cttgggtgtgggcatcagg	SEQ ID NO:76
HPA-2bb CR	tgggtgtgggcatcaggag	SEQ ID NO:77
HPA-2bc CR	ggtgtgggcatcaggagcc	SEQ ID NO:78
HPA-2bd CR	tgtgggcatcaggagccct	SEQ ID NO:79
HPA-2be CR	gggcatcaggagccctgg	SEQ ID NO:80
HPA3-allele a:
HPA-3aa	ccatccccagcccctccc	SEQ ID NO:81
HPA-3ab	gcccatccccagcccctc	SEQ ID NO:82
HPA-3ac	ctgcccatccccagcccc	SEQ ID NO:83
HPA-3ad1	gctgcccatccccagccc	SEQ ID NO:84
HPA-3ad	ggctgcccatccccagcc	SEQ ID NO:85
HPA-3ad2	gggctgcccatccccagc	SEQ ID NO:86
HPA-3ae	ggggctgcccatcccca	SEQ ID NO:87
HPA-3aa CR	gggaggggctggggatgg	SEQ ID NO:88
HPA-3ab CR	gaggggctggggatgggc	SEQ ID NO:89
HPA-3ac CR	ggggctggggatgggcag	SEQ ID NO:90
HPA-3ad1 CR	gggctggggatgggcagc	SEQ ID NO:91
HPA-3ad CR	ggctggggatgggcagcc	SEQ ID NO:92
HPA-3ad2 CR	gctggggatgggcagccc	SEQ ID NO:93
HPA-3ae CR	tgggatgggcagcccc	SEQ ID NO:94
HPA3-allele b:
HPA-3ba	ccagccccagcccctcc	SEQ ID NO:95
HPA-3bb	gcccagccccagcccct	SEQ ID NO:96
HPA-3bc	ctgcccagccccagccc	SEQ ID NO:97
HPA-3bd1	gctgcccagccccagcc	SEQ ID NO:98
HPA-3bd	ggctgcccagcccagc	SEQ ID NO:99
HPA-3bd2	gggctgcccagccccag	SEQ ID NO:100
HPA-3be	ggggctgcccagcccca	SEQ ID NO:101
HPA-3ba CR	ggaggggctggggctgg	SEQ ID NO:102
HPA-3bb CR	aggggctggggctgggc	SEQ ID NO:103
HPA-3bc CR	gggctggggctgggcag	SEQ ID NO:104
HPA-3bd1 CR	ggctggggctgggcagc	SEQ ID NO:105
HPA-3bd CR	gctggggctgggcagcc	SEQ ID NO:106
HPA-3bd2 CR	ctggggctgggcagccc	SEQ ID NO:107
HPA-3be CR	tggggctgggcagcccc	SEQ ID NO:108
HPA4-allele a:
HPA-4aa	gccacccagatgcgaaag	SEQ ID NO:109
HPA-4ab	cacccagatgcgaaagct	SEQ ID NO:110
HPA-4ac	cccagatgcgaaagctca	SEQ ID NO:111
HPA-4ad	cagatgcgaaagctcacc	SEQ ID NO:112
HPA-4ae	gatgcgaaagctcaccag	SEQ ID NO:113
HPA-4aa CR	ctggtgagctttcgcatc	SEQ ID NO:114
HPA-4ab CR	ggtgagctttcgcatctg	SEQ ID NO:115
HPA-4ac CR	tgagctttcgcatctggg	SEQ ID NO:116
HPA-4ad CR	agctttcgcatctgggtg	SEQ ID NO:117
HPA-4ae CR	ctttcgcatctgggtggc	SEQ ID NO:118
HPA4-allele b:
HPA-4ba	gccacccagatgcaaaag	SEQ ID NO:119
HPA-4bb	ccacccagatgcaaaagct	SEQ ID NO:120
HPA-4bc	acccagatgcaaaagctcac	SEQ ID NO:121
HPA-4bd	cagatgcaaaagctcacca	SEQ ID NO:122
HPA-4be	gatgcaaaagctcaccagtaa	SEQ ID NO:123
HPA-4ba CR	ttactggtgagcttttgcatc	SEQ ID NO:124
HPA-4bb CR	tggtgagcttttgcatctg	SEQ ID NO:125
HPA-4bc CR	gtgagcttttgcatctgggt	SEQ ID NO:126
HPA-4bd CR	agcttttgcatctgggtgg	SEQ ID NO:127
HPA-4be CR	cttttgcatctgggtggc	SEQ ID NO:128
HPA5-allele a:
HPA-5aa	gagtctacctgtttactatcaaagagg	SEQ ID NO:129
HPA-5ab	agtctacctgttactatcaaagaggta	SEQ ID NO:130
HPA-5ac	gtctacctgtttactatcaaagaggtaa	SEQ ID NO:131
HPA-5ad	ctacctgtttactatcaaagaggtaaaa	SEQ ID NO:132
HPA-5ae	acctgtttactatcaaagaggtaaaaa	SEQ ID NO:133
HPA-5aa CR	tttttacctctttgatagtaaacaggt	SEQ ID NO:134
HPA-5ab CR	ttttacctctttgatagtaaacaggtag	SEQ ID NO:135
HPA-5ac CR	ttacctctttgatagtaaacaggtagac	SEQ ID NO:136
HPA-5ad CR	tacctctttgatagtaaacaggtagact	SEQ ID NO:137
HPA-5ae CR	cctctttgatagtaaacaggtagactc	SEQ ID NO:138
HPA5-allele b:
HPA-5ba	gagtctacctgtttactatcaaaaagg	SEQ ID NO:139
HPA-5bb	agtctacctgtttactatcaaaaaggta	SEQ ID NO:140
HPA-5bc	gtctacctgtttactatcaaaaaggtaa	SEQ ID NO:141
HPA-5ad	ctacctgtttactatcaaaaaggtaaaa	SEQ ID NO:142
HPA-5be	acctgtttactatcaaaaaggtaaaaa	SEQ ID NO:143
HPA-5ba CR	tttttacctttttgatagtaaacaggt	SEQ ID NO:144
HPA-5bb CR	ttttacctttttgatagtaaacaggtag	SEQ ID NO:145
HPA-5bc CR	ttacctttttgatagtaaacaggtagac	SEQ ID NO:146
HPA-5bd CR	tacctttttgatagtaaacaggtagact	SEQ ID NO:147
HPA-5be CR	cctttttgatagtaaacaggtagactc	SEQ ID NO:148
HPA15-allele a:
Gov-aa	ttattatcttgacttcagttacaggattt	SEQ ID NO:149
Gov-ab	tcttgacttcagttacaggatttacc	SEQ ID NO:150
Gov-ac	tgacttcagttacaggatttaccaa	SEQ ID NO:151
Gov-ad	cttcagttacaggatttaccaagaat	SEQ ID NO:152
Gov-ae	cagttacaggatttaccaagaatttg	SEQ ID NO:153
Gov-aa CR	caaattcttggtaaatcctgtaactg	SEQ ID NO:154
Gov-ab CR	attcttggtaaatcctgtaactgaag	SEQ ID NO:155
Gov-ac CR	tggtaaatcctgtaactgaagtcaa	SEQ ID NO:156
Gov-ad CR	ggtaaatcctgtaactgaagtcaaga	SEQ ID NO:157
Gov-ae CR	aaatcctgtaactgaagtcaagataataa	SEQ ID NO:158
HPA15-allele b:
Gov-ba	tatcttgacttcagttccaggatt	SEQ ID NO:159
Gov-bb	cttgacttcagttccaggatttac	SEQ ID NO:160
Gov-bc	gacttcagttccaggatttacca	SEQ ID NO:161
Gov-bd	ttcagttccaggatttaccaag	SEQ ID NO:162
Gov-be	cagttccaggatttaccaagaatt	SEQ ID NO:163
Gov-ba CR	aattcttggtaaatcctggaactg	SEQ ID NO:164
Gov-bb CR	cttggtaaatcctggaactgaa	SEQ ID NO:165
Gov-bc CR	ggtaaatcctggaactgaagtca	SEQ ID NO:166
Gov-bd CR	gtaaatcctggaactgaagtcaag	SEQ ID NO:167
Gov-be CR	aatcctggaactgaagtcaagata	SEQ ID NO:168
Colton-a-allele:
Co.a.1	aacaaccagacggcggt	SEQ ID NO:191
Co.a.2	accagacggcggtccag	SEQ ID NO:192
Co.a.3	cagacggcggtccagga	SEQ ID NO:193
Co.a.4	gacggcggtccaggacaa	SEQ ID NO:194
Co.a.5	cggcggtccaggacaac	SEQ ID NO:195
Co.a.1.cr	gttgtcctggaccgccgt	SEQ ID NO:196
Co.a.2.cr	tcctggaccgccgtctg	SEQ ID NO:197
Co.a.3.cr	ctggaccgccgtctggt	SEQ ID NO:198
Co.a.4.cr	ggaccgccgtctggttg	SEQ ID NO:199
Co.a.5.cr	accgccgtctggttgtt	SEQ ID NO:200
Colton-b-allele:
Co.b.1	ggaacaaccagacggtggt	SEQ ID NO:201
Co.b.2	acaaccagacggtggtccag	SEQ ID NO:202
Co.b.3	accagacggtggtccagga	SEQ ID NO:203
Co.b.4	gacggtggtccaggacaacg	SEQ ID NO:204
Co.b.5	cggtggtccaggacaacg	SEQ ID NO:205
Co.b.1.cr	cgttgtcctggaccaccgt	SEQ ID NO:206
Co.b.2.cr	ttgtcctggaccaccgtctg	SEQ ID NO:207
Co.b.3.cr	ctggaccaccgtctggttgt	SEQ ID NO:208
Co.b.4.cr	ggaccaccgtctggttgttc	SEQ ID NO:209
Co.b.5.cr	accaccgtctggttgttcc	SEQ ID NO:210
Diego-a-allele:
Di.a.1	gtgaagtccacgccggc	SEQ ID NO:211
Di.a.2	gaagtccacgccggcct	SEQ ID NO:212
Di.a.3	agtccacgccggcctcc	SEQ ID NO:213
Di.a.4	tccacgccggcctccct	SEQ ID NO:214
Di.a.5	acgccggcctccctggcc	SEQ ID NO:215
Di.a.1.cr	gccagggaggccggcgt	SEQ ID NO:216
Di.a.2.cr	agggaggccggcgtgga	SEQ ID NO:217
Di.a.3.cr	ggaggccggcgtggact	SEQ ID NO:218
Di.a.4.cr	aggccggcgtggacttc	SEQ ID NO:219
Di.a.5.cr	ggccggcgtggacttca	SEQ ID NO:220
Diego-b-allele:
Di.b.1	ggtgaagtccacgctggc	SEQ ID NO:221
Di.b.2	gtgaagtccacgctggcct	SEQ ID NO:222
Di.b.3	tgaagtccacgctggcctcc	SEQ ID NO:223
Di.b.4	gaagtccacgctggcctccct	SEQ ID NO:224
Di.b.5	acgctggcctccctggccc	SEQ ID NO:225
Di.b.1.cr	ggccagggaggccagcgt	SEQ ID NO:226
Di.b.2.cr	cagggaggccagcgtgga	SEQ ID NO:227
Di.b.3.cr	agggaggccagcgtggact	SEQ ID NO:228
Di.b.4.cr	ggaggccagcgtggacttc	SEQ ID NO:229
Di.b.5.cr	ggccagcgtggacttcacc	SEQ ID NO:230
Diego Wr-a-allele:
Wr.a.1	tgggcttgcgttccaagt	SEQ ID NO:231
Wr.a.2	ggcttgcgttccaagtttc	SEQ ID NO:232
Wr.a.3	ttgcgttccaagtttccca	SEQ ID NO:233
Wr.a.4	cgttccaagtttcccatct	SEQ ID NO:234
Wr.a.5	tccaagtttcccatctgga	SEQ ID NO:235
Wr.a.1 CR	tccagatgggaaacttgga	SEQ ID NO:236
Wr.a.2 CR	agatgggaaacttggaacg	SEQ ID NO:237
Wr.a.3 CR	tgggaaacttggaacgcaa	SEQ ID NO:238
Wr.a.4 CR	gaaacttggaacgcaagcc	SEQ ID NO:239
Wr.a.5 CR	acttggaacgcaagccca	SEQ ID NO:240
Diego Wr-b-allele:2
Wr.b.1	gggcttgcgttccgagtt	SEQ ID NO:241
Wr.b.2	gcttgcgttccgagtttc	SEQ ID NO:242
Wr.b.3	ttgcgttccgagtttccc	SEQ ID NO:243
Wr.b.4	cgttccgagtttcccatc	SEQ ID NO:244
Wr.b.5	tccgagtttcccatctgg	SEQ ID NO:245
Wr.b.1 CR	ccagatgggaaactcgga	SEQ ID NO:246
Wr.b.2 CR	gatgggaaactcggaacg	SEQ ID NO:247
Wr.b.3 CR	gggaaactcggaacgcaa	SEQ ID NO:248
Wr.b.4 CR	gaaactcggaacgcaagc	SEQ ID NO:249
Wr.b.5 CR	aactcggaacgcaagccc	SEQ ID NO:250
Dombrock-a-allele:
Do.a.1	taccacccaagaggaaact	SEQ ID NO:251
Do.a.2	ccacccaagaggaaactgg	SEQ ID NO:252
Do.a.3	acccaagaggaaactggttg	SEQ ID NO:253
Do.a.4	caagaggaaactggttgca	SEQ ID NO:254
Do.a.5	aggaaactggttgcagttga	SEQ ID NO:255
Do a 6 cr	ctcaactgcaaccagtttcc	SEQ ID NO:256
Do a 7 cr	caactgcaaccagtttcctc	SEQ ID NO:257
Do a 8 cr	tgcaaccagtttcctcttgg	SEQ ID NO:258
Do a 9 cr	accagtttcctcttgggtgg	SEQ ID NO:259
Do a 10 cr	cagtttcctcttgggtggta	SEQ ID NO:260
Dombrock-b-allele:
Do.b.1	taccacccaagaggagact	SEQ ID NO:261
Do.b.2	ccacccaagaggagactgg	SEQ ID NO:262
Do.b.3	acccaagaggagactggttg	SEQ ID NO:263
Do.b.4	caagaggagactggttgca	SEQ ID NO:264
Do.b.5	aggagactggttgcagttga	SEQ ID NO:265
Do b 6 cr	ctcaactgcaaccagtctcc	SEQ ID NO:266
Do b 7 cr	caactgcaaccagtctcctc	SEQ ID NO:267
Do b 8 cr	tgcaaccagtctcctcttgg	SEQ ID NO:268
Do b 9 cr	accagtctcctcttgggtgg	SEQ ID NO:269
Do b 10 cr	cagtctcctcttgggtggt	SEQ ID NO:270
Dombrock-Joseph(a) positive allele:
Jo.a.pos.1	ccccagaacatgactaccac	SEQ ID NO:271
Jo.a.pos.2	ccagaacatgactaccacaca	SEQ ID NO:272
Jo.a.pos.3	agaacatgactaccacacacgc	SEQ ID NO:273
Jo.a.pos.4	catgactaccacacacgctgt	SEQ ID NO:274
Jo.a.pos.5	actaccacacacgctgtgg	SEQ ID NO:275
Jo.a.pos.1.cr	gccacagcgtgtgtggtagt	SEQ ID NO:276
Jo.a.pos.2.cr	acagcgtgtgtggtagtcatg	SEQ ID NO:277
Jo.a.pos.3.cr	agcgtgtgtggtagtcatgtt	SEQ ID NO:278
Jo.a.pos.4.cr	cgtgtgtggtagtcatgttctg	SEQ ID NO:279
Jo.a.pos.5.cr	gtggtagtcatgttctgggg	SEQ ID NO:280
Dombrock-Joseph(a) negative allele:
Jo.a.neg.1	ctaccccagaacatgactatcac	SEQ ID NO:281
Jo.a.neg.2	cccagaacatgactatcacaca	SEQ ID NO:282
Jo.a.neg.3	cagaacatgactatcacacacgc	SEQ ID NO:283
Jo.a.neg.4	catgactatcacacacgctgtg	SEQ ID NO:284
Jo.a.neg.5	actatcacacacgctgtggc	SEQ ID NO:285
Jo.a.neg.1.cr	aatagccacagcgtgtgtgatagt	SEQ ID NO:286
Jo.a.neg.2.cr	cacagcgtgtgtgatagtcatg	SEQ ID NO:287
Jo.a.neg.3.cr	cagcgtgtgtgatagtcatgtt	SEQ ID NO:288
Jo.a.neg.4.cr	cgtgtgtgatagtcatgttctgg	SEQ ID NO:289
Jo.a.neg.5.cr	gtgatagtcatgttctggggtag	SEQ ID NO:290
Duffy-a-allele:
Fy.A.1	ccagatggagactatggtgcc	SEQ ID NO:291
Fy.A.2	atggagactatggtgccaac	SEQ ID NO:292
Fy.A.3	ggagactatggtgccaacctg	SEQ ID NO:293
Fy.A.4	gactatggtgccaacctgga	SEQ ID NO:294
Fy.A.5	tatggtgccaacctggaag	SEQ ID NO:295
Fy.A.1.cr	cttccaggttggcaccata	SEQ ID NO:296
Fy.A.2.cr	tccaggttggcaccatagtc	SEQ ID NO:297
Fy.A.3.cr	aggttggcaccatagtctcc	SEQ ID NO:298
Fy.A.4.cr	gttggcaccatagtctccat	SEQ ID NO:299
Fy.A.5.cr	gcaccatagtctccatctgg	SEQ ID NO:300
Duffy-b-allele:
Fy.B.1	cccagatggagactatgatgcc	SEQ ID NO:301
Fy.B.2	gatggagactatgatgccaac	SEQ ID NO:302
Fy.B.3	tggagactatgatgccaacctg	SEQ ID NO:303
Fy.B.4	gactatgatgccaacctggaa	SEQ ID NO:304
Fy.B.5	tatgatgccaacctggaagc	SEQ ID NO:305
Fy.B.1.cr	gcttccaggttggcatcata	SEQ ID NO:306
Fy.B.2.cr	ttccaggttggcatcatagtc	SEQ ID NO:307
Fy.B.3.cr	caggttggcatcatagtctcc	SEQ ID NO:308
Fy.B.4.cr	gttggcatcatagtctccatc	SEQ ID NO:309
Fy.B.5.cr	gcatcatagtctccatctggg	SEQ ID NO:310
Duffy GATAbox-normal-allele:
Fy.GATA.normal.1	agtccttggctcttatcttg	SEQ ID NO:311
Fy.GATA.normal.2	agtccttggctcttatcttgga	SEQ ID NO:312
Fy.GATA.normal.3	cttggctcttatcttggaagc	SEQ ID NO:313
Fy.GATA.normal.4	gctcttatcttggaagcacagg	SEQ ID NO:314
Fy.GATA.normal.5	cttatcttggaagcacaggcgc	SEQ ID NO:315
Fy.GATA.normal.1.cr	gcctgtgcttccaagataag	SEQ ID NO:316
Fy.GATA.normal.2.cr	tgtgcttccaagataagagc	SEQ ID NO:317
Fy.GATA.normal.3.cr	tgcttccaagataagagcca	SEQ ID NO:318
Fy.GATA.normal.4.cr	ttccaagataagagccaagga	SEQ ID NO:319
Fy.GATA.normal.5.cr	caagataagagccaaggact	SEQ ID NO:320
Duffy GATAbox-mutation-allele:
Fy.GATA.mut.1	tccttggctcttaccttg	SEQ ID NO:321
Fy.GATA.mut.2	tccttggctcttaccttgga	SEQ ID NO:322
Fy.GATA.mut.3	tggctcttaccttggaagc	SEQ ID NO:323
Fy.GATA.mut.4	gctcttaccttggaagcacag	SEQ ID NO:324
Fy.GATA.mut.5	cttaccttggaagcacaggcg	SEQ ID NO:325
Fy.GATA.mut.1.cr	cctgtgcttccaaggtaag	SEQ ID NO:326
Fy.GATA.mut.2.cr	gtgcttccaaggtaagagc	SEQ ID NO:327
Fy.GATA.mut.3.cr	gcttccaaggtaagagcca	SEQ ID NO:328
Fy.GATA.mut.4.cr	ttccaaggtaagagccaagg	SEQ ID NO:329
Fy.GATA.mut.5.cr	caaggtaagagccaagga	SEQ ID NO:330
Duffy Fyx-normal-allele:
Fy.X.(b).1	ttttcagacctctcttccgct	SEQ ID NO:331
Fy.X.(b).2	agacctctcttccgctggc	SEQ ID NO:332
Fy.X.(b).3	ctctcttccgctggcagc	SEQ ID NO:333
Fy.X.(b).4	ctcttccgctggcagctc	SEQ ID NO:334
Fy.X.(b).5	cttccgctggcagctctg	SEQ ID NO:335
Fy.X.(b).1.cr	cagagctgccagcggaa	SEQ ID NO:336
Fy.X.(b).2.cr	agctgccagcggaagag	SEQ ID NO:337
Fy.X.(b).3.cr	ctgccagcggaagagagg	SEQ ID NO:338
Fy.X.(b).4.cr	gccagcggaagagaggtc	SEQ ID NO:339
Fy.X.(b).5.cr	cagcggaagagaggtctg	SEQ ID NO:340
Duffy Fyx-mutation-allele:
Fy.X.1	gcttttcagacctctcttctgct	SEQ ID NO:341
Fy.X.2	tcagacctctcttctgctggc	SEQ ID NO:342
Fy.X.3	acctctcttctgctggcagc	SEQ ID NO:343
Fy.X.4	ctcttctgctggcagctctg	SEQ ID NO:344
Fy.X.5	cttctgctggcagctctgc	SEQ ID NO:345
Fy.X.1.cr	gcagagctgccagcagaa	SEQ ID NO:346
Fy.X.2.cr	gagctgccagcagaagagag	SEQ ID NO:347
Fy.X.3.cr	agctgccagcagaagagagg	SEQ ID NO:348
Fy.X.4.cr	gccagcagaagagaggtctg	SEQ ID NO:349
Fy.X.5.cr	cagcagaagagaggtctgaaa	SEQ ID NO:350
Kidd-allele-a:
Jk.a.1	cagccccatttgaggaca	SEQ ID NO:351
Jk.a.2	gccccatttgaggacatcta	SEQ ID NO:352
Jk.a.3	ccatttgaggacatctactttg	SEQ ID NO:353
Jk.a.4	atttgaggacatctactttgga	SEQ ID NO:354
Jk.a.5	gaggacatctactttggactct	SEQ ID NO:355
Jk.a.1.cr	cagagtccaaagtagatgtcctc	SEQ ID NO:356
Jk.a.2.cr	agtccaaagtagatgtcctcaaa	SEQ ID NO:357
Jk.a.3.cr	aaagtagatgtcctcaaatggg	SEQ ID NO:358
Jk.a.4.cr	tagatgtcctcaaatggggc	SEQ ID NO:359
Jk.a.5.cr	atgtcctcaaatggggctg	SEQ ID NO:360
Kidd-allele-b:
Jk.b.1	tcagccccatttgagaaca	SEQ ID NO:361
Jk.b.2	gccccatttgagaacatcta	SEQ ID NO:362
Jk.b.3	cccatttgagaacatctactttg	SEQ ID NO:363
Jk.b.4	atttgagaacatctactttggac	SEQ ID NO:364
Jk.b.5	gagaacatctactttggactctg	SEQ ID NO:365
Jk.b.1.cr	ccagagtccaaagtagatgttctc	SEQ ID NO:366
Jk.b.2.cr	agagtccaaagtagatgttctcaaa	SEQ ID NO:367
Jk.b.3.cr	ccaaagtagatgttctcaaatgg	SEQ ID NO:368
Jk.b.4.cr	agtagatgttctcaaatggggc	SEQ ID NO:369
Jk.b.5.cr	atgttctcaaatggggctga	SEQ ID NO:370
Kell-K1-allele:
KEL.1.1	tccttaaactttaaccgaatgct	SEQ ID NO:371
KEL.1.2	ttaaactttaaccgaatgctgaga	SEQ ID NO:372
KEL.1.3	aactttaaccgaatgctgagactt	SEQ ID NO:373
KEL.1.4	aaccgaatgctgagacttctg	SEQ ID NO:374
KEL.1.5	cgaatgctgagacttctgatgag	SEQ ID NO:375
KEL 1.6 CR	actcatcagaagtctcagcattc	SEQ ID NO:376
KEL 1.7 CR	tcagaagtctcagcattcggt	SEQ ID NO:377
KEL 1.8 CR	aagtctcagcattcggttaaag	SEQ ID NO:378
KEL 1.9 CR	tctcagcattcggttaaagtttaa	SEQ ID NO:379
KEL 1.10 CR	agcattcggttaaagtttaagga	SEQ ID NO:380
Kell-K2-allele:
KEL.2.1	ccttaaactttaaccgaacgct	SEQ ID NO:381
KEL.2.2	aactttaaccgaacgctgaga	SEQ ID NO:382
KEL.2.3	ctttaaccgaacgctgagactt	SEQ ID NO:383
KEL.2.4	aaccgaacgctgagacttct	SEQ ID NO:384
KEL.2.5	cgaacgctgagacttctgatg	SEQ ID NO:385
KEL 2.6 CR	tcatcagaagtctcagcgttc	SEQ ID NO:386
KEL 2.7 CR	cagaagtctcagcgttcggt	SEQ ID NO:387
KEL 2.8 CR	agtctcagcgttcggttaaag	SEQ ID NO:388
KEL 2.9 CR	tctcagcgttcggttaaagtt	SEQ ID NO:389
KEL 2.10 CR	agcgttcggttaaagtttaagg	SEQ ID NO:390
Kell-K3-allele:
KEL.3.1	aatctccatcacttcatggct	SEQ ID NO:391
KEL.3.2	tccatcacttcatggctgtt	SEQ ID NO:392
KEL.3.3	atcacttcatggctgttcca	SEQ ID NO:393
KEL.3.4	acttcatggctgttccagtt	SEQ ID NO:394
KEL.3.5	tcatggctgttccagtttc	SEQ ID NO:395
KEL.3.1.cr	agaaactggaacagccatgaa	SEQ ID NO:396
KEL.3.2.cr	aactggaacagccatgaagtg	SEQ ID NO:397
KEL.3.3.cr	tggaacagccatgaagtgatg	SEQ ID NO:398
KEL.3.4.cr	acagccatgaagtgatggag	SEQ ID NO:399
KEL.3.5.cr	gccatgaagtgatggagatt	SEQ ID NO:400
Kell-K4-allele:
KEL.4.1	tctccatcacttcacggct	SEQ ID NO:401
KEL.4.2	ccatcacttcacggctgtt	SEQ ID NO:402
KEL.4.3	cacttcacggctgttcca	SEQ ID NO:403
KEL.4.4	acttcacggctgttccag	SEQ ID NO:404
KEL.4.5	tcacggctgttccagttt	SEQ ID NO:405
KEL.4.1.cr	aaactggaacagccgtgaa	SEQ ID NO:406
KEL.4.2.cr	ctggaacagccgtgaagtg	SEQ ID NO:407
KEL.4.3.cr	ggaacagccgtgaagtgatg	SEQ ID NO:408
KEL.4.4.cr	acagccgtgaagtgatgg	SEQ ID NO:409
KEL.4.5.cr	gccgtgaagtgatggaga	SEQ ID NO:410
Kell-K6-allele:
KEL.6.1	tactgcctgggggctgccccgcc	SEQ ID NO:411
KEL.6.2	tgggggctgccccgcctgt	SEQ ID NO:412
KEL.6.3	gctgccccgcctgtgac	SEQ ID NO:413
KEL.6.4	ctgccccgcctgtgacaa	SEQ ID NO:414
KEL.6.5	gccccgcctgtgacaac	SEQ ID NO:415
KEL.6.1.cr	gttgtcacaggcggggc	SEQ ID NO:416
KEL.6.2.cr	ttgtcacaggcggggcag	SEQ ID NO:417
KEL.6.3.cr	tcacaggcggggcagccc	SEQ ID NO:418
KEL.6.4.cr	acaggcggggcagccccca	SEQ ID NO:419
KEL.6.5.cr	aggcggggcagccccca	SEQ ID NO:420
Kell-K7-allele:
KEL.7.1	actgcctgggggctgcctcgcc	SEQ ID NO:421
KEL.7.2	cctgggggctgcctcgcctgt	SEQ ID NO:422
KEL.7.3	ggctgcctcgcctgtgac	SEQ ID NO:423
KEL.7.4	ctgcctcgcctgtgacaacc	SEQ ID NO:424
KEL.7.5	gcctcgcctgtgacaacc	SEQ ID NO:425
KEL.7.1.cr	ggttgtcacaggcgaggc	SEQ ID NO:426
KEL.7.2.cr	ggttgtcacaggcgaggcag	SEQ ID NO:427
KEL.7.3.cr	ttgtcacaggcgaggcagccc	SEQ ID NO:428
KEL.7.4.cr	acaggcgaggcagcccccagg	SEQ ID NO:429
KEL.7.5.cr	aggcgaggcagcccccagg	SEQ ID NO:430
Lutheran a-allele:
Lu.a.1	ggagctcgcccccgcct	SEQ ID NO:431
Lu.a.2	gctcgcccccgcctagc	SEQ ID NO:432
Lu.a.3	cgcccccgcctagcctc	SEQ ID NO:433
Lu.a.4	cccccgcctagcctcgg	SEQ ID NO:434
Lu.a.5	cccgcctagcctcggct	SEQ ID NO:435
Lu.a.1 CR	agccgaggctaggcggg	SEQ ID NO:436
Lu.a.2 CR	ccgaggctaggcggggg	SEQ ID NO:437
Lu.a.3 CR	gaggctaggcgggggcg	SEQ ID NO:438
Lu.a.4 CR	gctaggcgggggcgagc	SEQ ID NO:439
Lu.a.5 CR	aggcgggggcgagctcc	SEQ ID NO:440
Lutheran b-allele:
Lu.b.1	gggagctcgcccccacct	SEQ ID NO:441
Lu.b.2	gagctcgcccccacctagc	SEQ ID NO:442
Lu.b.3	ctcgcccccacctagcctc	SEQ ID NO:443
Lu.b.4	cccccacctagcctcggct	SEQ ID NO:444
Lu.b.5	cccacctagcctcggctga	SEQ ID NO:445
Lu.b.1 CR	tcagccgaggctaggtggg	SEQ ID NO:446
Lu.b.2 CR	agccgaggctaggtggggg	SEQ ID NO:447
Lu.b.3 CR	gaggctaggtgggggcgag	SEQ ID NO:448
Lu.b.4 CR	gctaggtgggggcgagctc	SEQ ID NO:449
Lu.b.5 CR	aggtgggggcgagctccc	SEQ ID NO:450
MNS M-allele:
M.1	gtgagcatatcagcatcaag	SEQ ID NO:451
M.2	atcagcatcaagtaccactgg	SEQ ID NO:452
M.3	catcaagtaccactggtgtg	SEQ ID NO:453
M.4	taccactggtgtggcaatgc	SEQ ID NO:454
M.5	ctggtgtggcaatgcaca	SEQ ID NO:455
M.1.cr	tgtgcattgccacaccagt	SEQ ID NO:456
M.2.cr	gcattgccacaccagtggta	SEQ ID NO:457
M.3.cr	ccacaccagtggtacttgatg	SEQ ID NO:458
M.4.cr	accagtggtacttgatgct	SEQ ID NO:459
M.5.cr	cttgatgctgatatgctcac	SEQ ID NO:460
MNS N-allele:
N.1	tgtgagcatatcagcattaag	SEQ ID NO:461
N.2	atcagcattaagtaccactgagg	SEQ ID NO:462
N.3	cattaagtaccactgaggtgg	SEQ ID NO:463
N.4	accactgaggtggcaatgc	SEQ ID NO:464
N.5	ctgaggtggcaatgcacact	SEQ ID NO:465
N.1.cr	gtgtgcattgccacctcagt	SEQ ID NO:466
N.2.cr	gcattgccacctcagtggta	SEQ ID NO:467
N.3.cr	ccacctcagtggtacttaatgc	SEQ ID NO:468
N.4.cr	cctcagtggtacttaatgct	SEQ ID NO:469
N.5.cr	cttaatgctgatatgctcaca	SEQ ID NO:470
MNS S-allele:
big.S.1	tttgctttataggagaaatggga	SEQ ID NO:471
big.S.2	ctttataggagaaatgggaca	SEQ ID NO:472
big.S.3	ttataggagaaatgggacaacttg	SEQ ID NO:473
big.S.4	gagaaatgggacaacttgtcc	SEQ ID NO:474
big.S.5	aaatgggacaacttgtccatc	SEQ ID NO:475
big.S.1.cr	gatggacaagttgtcccattt	SEQ ID NO:476
big.S.2.cr	gacaagttgtcccatttctcc	SEQ ID NO:477
big.S.3.cr	aagttgtcccatttctcctata	SEQ ID NO:478
bigS.4.cr	tgtcccatttctcctataaagca	SEQ ID NO:479
big.S.5.cr	cccatttctcctataaagcaaaa	SEQ ID NO:480
MNS s-allele:
little.s.1	tgctttataggagaaacggga	SEQ ID NO:481
little.s.2	tttataggagaaacgggaca	SEQ ID NO:482
little.s.3	ggagaaacgggacaacttg	SEQ ID NO:483
little.s.4	gagaaacgggacaacttgtc	SEQ ID NO:484
little.s.5	aaacgggacaacttgtccat	SEQ ID NO:485
little.s.1.cr	tggacaagttgtcccgttt	SEQ ID NO:486
little.s.2.cr	acaagttgtcccgtttctcc	SEQ ID NO:487
little.s.3.cr	agttgtcccgtttctcctata	SEQ ID NO:488
little.s.4.cr	tgtcccgtttctcctataaagc	SEQ ID NO:489
little.s.5.cr	cccgtttctcctataaagca	SEQ ID NO:490
MNS U-positive-allele:
U.pos.1	ttgctgctctctttagctcc	SEQ ID NO:491
U.pos.2	ctctctttagctcctgtagtgat	SEQ ID NO:492
U.pos.3	agctcctgtagtgataatactca	SEQ ID NO:493
U.pos.4	gtagtgataatactcattatttttg	SEQ ID NO:494
U.pos.5	taatactcattatttttggggtg	SEQ ID NO:495
U.pos.1.cr	caccccaaaaataatgagtatta	SEQ ID NO:496
U.pos.2.cr	caaaaataatgagtattatcactaca	SEQ ID NO:497
U.pos.3.cr	agtattatcactacaggagctaaa	SEQ ID NO:498
U.pos.4.cr	atcactacaggagctaaagag	SEQ ID NO:499
U.pos.5.cr	gagctaaagagagcagcaaa	SEQ ID NO:500
MNS U-negative-allele:
U.neg.1	ttttgctgctctctttatctcc	SEQ ID NO:501
U.neg.2	gctctctttatctcctgtagagat	SEQ ID NO:502
U.neg.3	tatctcctgtagagataacactca	SEQ ID NO:503
U.neg.4	gtagagataacactcattattttt	SEQ ID NO:504
U.neg.5	taacactcattatttttggggt	SEQ ID NO:505
U.neg.1.cr	accccaaaaataatgagtgtta	SEQ ID NO:506
U.neg.2.cr	aaaaataatgagtgttatctctaca	SEQ ID NO:507
U.neg.3.cr	agtgttatctctacaggagataaa	SEQ ID NO:508
U.neg.4.cr	atctctacaggagataaagagag	SEQ ID NO:509
U.neg.5.cr	gagataaagagagcagcaaaatta	SEQ ID NO:510
Rhesus C-allele(307T):
Rh.big.C.1	tgagccagttcccttctgg	SEQ ID NO:511
Rh.big.C.2	gagccagttcccttctgg	SEQ ID NO:512
Rh.big.C.3	ctgagccagttcccttctg	SEQ ID NO:513
Rh.big.C.4	ccttctgggaaggtggtc	SEQ ID NO:514
Rh.big.C.5	ccttctgggaaggtggtca	SEQ ID NO:515
Rh.big.C.1.cr	tgaccaccttcccagaagg	SEQ ID NO:516
Rh.big.C.2.cr	gaccaccttcccagaagg	SEQ ID NO:517
Rh.big.C.3.cr	ccagaagggaactggctc	SEQ ID NO:518
Rh.big.C.4.cr	ccagaagggaactggctca	SEQ ID NO:519
Rh.big.C.5.cr	cagaagggaactggctcag	SEQ ID NO:520
Rhesus c-allele(307C):
Rh.little.c.1	gagccagttccctcctgg	SEQ ID NO:521
Rh.little.c.2	agccagttccctcctgg	SEQ ID NO:522
Rh.little.c.3	tgagccagttccctcctg	SEQ ID NO:523
Rh.little.c.4	cctcctgggaaggtggt	SEQ ID NO:524
Rh.little.c.5	cctcctgggaaggtggtc	SEQ ID NO:525
Rh.little.c.1.cr	gaccaccttcccaggagg	SEQ ID NO:526
Rh.little.c.2.cr	accaccttcccaggagg	SEQ ID NO:527
Rh.little.c.3.cr	ccaggagggaactggct	SEQ ID NO:528
Rh.little.c.4.cr	ccaggagggaactggctc	SEQ ID NO:529
Rh.little.c.5.cr	caggagggaactggctca	SEQ ID NO:530
Rhesus BigC-intron2-specific-insert-allele:
RhC.intron.2.1	agggtgccctttgtcacttc	SEQ ID NO:531
RhC.intron.2.2	gccctttgtcacttcccagt	SEQ ID NO:532
RhC.intron.2.3	cctttgtcacttcccagtgg	SEQ ID NO:533
RhC.intron.2.4	ttgtcacttcccagtggtacaa	SEQ ID NO:534
RhC.intron.2.5	tcacttcccagtggtacaatca	SEQ ID NO:535
RhC.intron.2.1.cr	gaagtgacaaagggcaccct	SEQ ID NO:536
RhC.intron.2.2.cr	actgggaagtgacaaagggc	SEQ ID NO:537
RhC.intron.2.3.cr	ccactgggaagtgacaaagg	SEQ ID NO:538
RhC.intron.2.4.cr	tgtaccactgggaagtgacaaa	SEQ ID NO:539
RhC.intron.2.5.cr	tgattgtaccactgggaagtga	SEQ ID NO:540
Rhesus BigC-intron2-specific-insert-negative-allele:

The setup of the PCR is chosen in such a way that a PCR product will be formed only, when the bigC-intron2-specific-insert is present. The antitags (used for background subtraction) are used to calculate the ratio for genotyping.

Rhesus E-allele:
Rh.big.E.1	gccaagtgtcaactctcctct	SEQ ID NO:541
Rh.big.E.2	caagtgtcaactctcctctgct	SEQ ID NO:542
Rh.big.E.3	gtgtcaactctcctctgctgag	SEQ ID NO:543
Rh.big.E.4	caactctcctctgctgagaagtc	SEQ ID NO:544
Rh.big.E.5	tctcctctgctgagaagtcc	SEQ ID NO:545
Rh.big.E.1.cr	ggacttctcagcagaggagag	SEQ ID NO:546
Rh.big.E.2.cr	cttctcagcagaggagagttga	SEQ ID NO:547
Rh.big.E.3.cr	ctcagcagaggagagttgacac	SEQ ID NO:548
Rh.big.E.4.cr	gcagaggagagttgacacttg	SEQ ID NO:549
Rh.big.E.5.cr	gaggagagttgacacttggc	SEQ ID NO:550
Rhesus e-allele:
Rh.little.e.1	gccaagtgtcaactctgctct	SEQ ID NO:551
Rh.little.e.2	caagtgtcaactctgctctgct	SEQ ID NO:552
Rh.little.e.3	tgtcaactctgctctgctgag	SEQ ID NO:553
Rh.little.e.4	caactctgctctgctgagaag	SEQ ID NO:554
Rh.little.e.5	tctgctctgctgagaagtcc	SEQ ID NO:555
Rh.little.e.1.cr	ggacttctcagcagagcagag	SEQ ID NO:556
Rh.little.e.2.cr	ttctcagcagagcagagttga	SEQ ID NO:557
Rh.little.e.3.cr	tcagcagagcagagttgacac	SEQ ID NO:558
Rh.little.e.4.cr	gcagagcagagttgacacttg	SEQ ID NO:559
Rh.little.e.5.cr	gagcagagttgacacttggc	SEQ ID NO:560
Rhesus RHD-allele:
RhD 1	ttccccacagctccatcat	SEQ ID NO:561
RhD 2	acagctccatcatgggctac	SEQ ID NO:562
RhD 3	tccatcatgggctacaacttc	SEQ ID NO:563
RbD 4	tcatgggctacaacttcagct	SEQ ID NO:564
RhD 5	gggctacaacttcagcttgct	SEQ ID NO:565
RhD cr 1	agcaagctgaagttgtagccc	SEQ ID NO:566
RbD cr 2	agctgaagttgtagcccatga	SEQ ID NO:567
RbD cr 3	gaagttgtagcccatgatgg	SEQ ID NO:568
RhD cr 4	gcccatgatggagctgt	SEQ ID NO:569
RhD cr 5	tgatggagctgtggggaa	SEQ ID NO:570
Rhesus RHD-negative-allele:

The setup of the PCR is chosen in such a way that a PCR product will be formed only, when the RHD gene is present. The antitags (used for background subtraction) are used to calculate the ratio for genotyping.

Rhesus r's-allele:
r′s.T.1	ggaaggtcaacttggtgca	SEQ ID NO:571
r′s.T.2	ggaaggtcaacttggtgcagt	SEQ ID NO:572
r′s.T.3	caacttggtgcagttggtg	SEQ ID NO:573
r′s.T.4	acttggtgcagttggtggt	SEQ ID NO:574
r′s.T.5	ttggtgcagttggtggtgat	SEQ ID NO:575
r′s.T.1.cr	catcaccaccaactgcacca	SEQ ID NO:576
r′s.T.2.cr	caccaccaactgcaccaagt	SEQ ID NO:577
r′s.T.3.cr	accaactgcaccaagttgac	SEQ ID NO:578
r′s.T.4.cr	actgcaccaagttgaccttcc	SEQ ID NO:579
r′s.T.5.cr	tgcaccaagttgaccttcc	SEQ ID NO:580
Rhesus r's-negative-allele:

The setup of the PCR is chosen in such a way that a PCR product will be formed only, when the r's gene is present. The antitags (used for background subtraction) are used to calculate the ratio for genotyping. Rhesus DVI-Allele:

The setup of the PCR is chosen in such a way that a PCR product will be formed only, when a normal RHD gene is present. The antitags (used for background subtraction) are used to calculate the ratio for genotyping.

DVI-probes for presence normal RHD allele:
Rh.DVI.1	atttcaaccctcttggcctt	SEQ ID NO:581
Rh.DVI.2	aaccctcttggcctttgttt	SEQ ID NO:582
Rh.DVI.3	tcttggcctttgtttccttg	SEQ ID NO:583
Rh.DVI.4	ggtatcagcttgagagctcg	SEQ ID NO:584
Rh.DVI.5	atcagcttgagagctcggag	SEQ ID NO:585
Rh.DVI.1.cr	aaggccaagagggttgaaat	SEQ ID NO:586
Rh.DVI.2.cr	aaacaaaggccaagagggtt	SEQ ID NO:587
Rh.DVI.3.cr	caaggaaacaaaggccaaga	SEQ ID NO:588
Rh.DVI.4.cr	cgagctctcaagctgatacc	SEQ ID NO:589
Rh.DVI.5.cr	ctccgagctctcaagctgat	SEQ ID NO:590
Rhesus RHD-Pseudogene-mutation-allele:
RhD Y 1	tttctttgcagacttaggtgc	SEQ ID NO:591
RhD Y 2	ctttgcagacttaggtgcaca	SEQ ID NO:592
RhD Y 3	tttgcagacttaggtgcacagt	SEQ ID NO:593
RhD Y 4	acttaggtgcacagtgcgg	SEQ ID NO:594
RhD Y 5	cttaggtgcacagtgcggt	SEQ ID NO:595
RhD Y cr 1	caccgcactgtgcacctaa	SEQ ID NO:596
RhD Y cr 2	ccgcactgtgcacctaagtc	SEQ ID NO:597
RhD Y cr 3	gcactgtgcacctaagtctgc	SEQ ID NO:598
RhD Y cr 4	tgcacctaagtctgcaaaga	SEQ ID NO:599
RhD Y cr 5	cacctaagtctgcaaagaaatagcg	SEQ ID NO:600
Rhesus RHD-Pseudogene-normal-allele:
RhD non Y 1	ctatttctttgcagacttatgtgc	SEQ ID NO:601
RhD non Y 2	tctttgcagacttatgtgcaca	SEQ ID NO:602
RhD non Y 3	ttgcagacttatgtgcacagtg	SEQ ID NO:603
RhD non Y 4	acttatgtgcacagtgcggt	SEQ ID NO:604
RhD non Y 5	cttatgtgcacagtgcggtg	SEQ ID NO:605
RhD non Y cr 1	acaccgcactgtgcacataa	SEQ ID NO:606
RhD non Y cr 2	accgcactgtgcacataagtc	SEQ ID NO:607
RhD non Y cr 3	gcactgtgcacataagtctgc	SEQ ID NO:608
RhD non Y cr 4	tgtgcacataagtctgcaaag	SEQ ID NO:609
RhD non Y cr 5	cacataagtctgcaaagaaatagcg	SEQ ID NO:610
Yt-a-allele:
Yt.a.1	gcgggagacttccacgg	SEQ ID NO:611
Yt.a.2	gggagacttccacggcct	SEQ ID NO:612
Yt.a.3	gagacttccacggcctgc	SEQ ID NO:613
Yt.a.4	gacttccacggcctgca	SEQ ID NO:614
Yt.a.5	ttccacggcctgcaggta	SEQ ID NO:615
Yt.a.1 CR	tacctgcaggccgtggaa	SEQ ID NO:616
Yt.a.2 CR	tgcaggccgtggaagtc	SEQ ID NO:617
Yt.a.3 CR	gcaggccgtggaagtctc	SEQ ID NO:618
Yt.a.4 CR	aggccgtggaagtctccc	SEQ ID NO:619
Yt.a.5 CR	ccgtggaagtctcccgc	SEQ ID NO:620
Yt-b-allele:
Yt.b.1	gcgggagacttcaacggc	SEQ ID NO:621
Yt.b.2	gggagacttcaacggcctg	SEQ ID NO:622
Yt.b.3	gagacttcaacggcctgca	SEQ ID NO:623
Yt.b.4	gacttcaacggcctgcag	SEQ ID NO:624
Yt.b.5	ttcaacggcctgcaggtaa	SEQ ID NO:625
Yt.b.1 CR	ttacctgcaggccgttgaa	SEQ ID NO:626
Yt.b.2 CR	ctgcaggccgttgaagtc	SEQ ID NO:627
Yt.b.3 CR	tgcaggccgttgaagtctc	SEQ ID NO:628
Yt.b.4 CR	caggccgttgaagtctccc	SEQ ID NO:629
Yt.b.5 CR	gccgttgaagtctcccgc	SEQ ID NO:630

Using the available software programs, it would be possible for a skilled person to design probes for other blood cell antigen alleles.

The DNA array may be a limited array comprising for example only 72 different probes, but preferably it comprises substantially all probes shown in the above list.

In order to facilitate binding of the probes to the array support and to allow sufficient access to the amplification products, the probe oligos will normally be provided with a linker molecule and a reactive group, preferably at the 5′-end of the oligo, for binding to the array support. All of this is well known to the skilled person and only by way of example it is mentioned that a suitable linker is C6 (hexamethylene, i.e. —(CH₂)₆—) and a suitable reactive group is amino.

Array Support

The probes are located on a suitable support to which they are usually attached by chemical means. The support may be a glass slide or any other suitable support, such as plastic, silicon or porous metal oxide. Usually, the support will be pretreated to improve binding of the oligos, for example pretreated by coating with poly-L-lysine, aminosilane, aldehyde or epoxy. To achieve binding of the probes, various techniques may be used, such as UV irradiation, nonspecific electrostatic absorption, covalent attachment via 5′amino or phosphate group using carbodiimide (EDC) chemistry, etc.

To avoid non-specific hybridization it will usually be preferable to incubate the array support carrying the probes thereon with prehybridization solution, as is well known to a skilled person, and to prepare the probes for hybridization it will be preferred to denature them, for example by heating at about 80° C. or higher.

Controls

In order to allow correction for background it is preferred to include some probes with a sequence not occurring in the DNA of the donor and which may be expected not to bind by hybridization to amplification products. The signal measured for allele-specific probe spots may be corrected by subtracting the signal measured for these background control spots. Suitable examples of such background correction probes or antitags are the following probes a3, a9, a17, a23, a27, a33, a35, a38, a42 and a43:

	a3	cagaccataagcacaggcgt,	SEQ ID NO:631
	a9	gctcgtccacagtgcgttat,	SEQ ID NO:632
	a17	cggcgttcaagcaaaccgaa,	SEQ ID NO:633
	a23	gacatatagctccactcaga,	SEQ ID NO:634
	a27	tagggtactgatgagcactc,	SEQ ID NO:635
	a33	tcagccctatcgcaggatgt,	SEQ ID NO:169
	a35	gagacacttgacagtagcca,	SEQ ID NO:170
	a38	ggcagggcacctcagtttat,	SEQ ID NO:171
	a42	tcaccagccagactgtgtag,	SEQ ID NO:172
	a43	cttcacgcaagttgtccaga.	SEQ ID NO:173

Further, it may be advisable to include at least one positive control probe. This probe should be the complement of a labeled positive control oligomer added to the products of the amplification. For example, the positive control probe may be the following oligo CS05:

CS05

gtcctgacttctagctcatg,

SEQ ID NO:174

which is capable of hybridizing to its labelled complement catgagctagaagtcaggac which is added to the mixture of amplification products before the mixture is applied onto the DNA array. Hybridization Conditions

Hybridization of products of the amplification to the probes in the DNA array is usually carried out at a temperature of between 50 and 65° C., preferably at about 56° C., for a sufficient time, such as for 15 minutes to 10 hours, more preferably for 30 minutes to 4 hours.

Array Configuration

The configuration of the array is fully arbitrary. Usually, one array support will carry a large number of spots present in several blocks that may be identical (preferred) or different from each other. Each spot contains one probe and it is preferred that similar probes are spotted as distant from each other as possible. It is preferable that each block contains one spot of each of the allele-specific probes, and in addition some control spots. Thus, one block may comprise 128 allele-specific spots, 5 background controls and a positive control, in total 134 spots.

Measuring the Fluorescence Signal

The binding of labelled amplification products to certain spots is determined on the basis of the fluorescence signal emitted by these spots. The signal can be measured with a photomultiplier tube, scanning laser systems or any other apparatus and technique known to the skilled person.

Evaluation of the Resulting Data

To evaluate the resulting data as to the blood cell antigen genotype of the donor of the DNA, the fluorescence signal intensities may be converted using Genepix Pro 5.0 (Axon Instruments Inc.) to signal values which can be analyzed further, for example in Excel.

EXAMPLES

In the Examples below, that only serve to illustrate the invention without limiting it in any way, the concept of genotyping blood cell antigens by the present combination of a particular multiplex PCR and a DNA microarray of allele-specific probes is tested in Example 1 on the biallelic Human Platelet Antigen systems HPA-1 through 5 and Gov. A blind panel of 58 donor samples was genotyped for these 6 HPA systems and only one discrepancy was found in one HPA system. The discrepancy can be overcome by adjusting the scoring criteria and validating the new format. Then, in Example 2, it was shown that the multiplex PCR of this invention worked well with all primers of Table II together.

Example 1

Sequences and Tm values of the probes spotted on poly-Lysine-coated glass slide are shown in Table I. The SNPs of HPA-3 and -5 are located in a GC-rich and -poor area, respectively. Therefore, these probes are shorter or longer than the other oligonucleotides, and have a Tm outside the 60-65° C. range. The probes, dissolved at a concentration of 50 μM in 0.4 M NaHCO₃ (pH 9.4), were spotted on poly-L-lysine coated glass slides by the Omnigrid 100® microarrayer (Genemachines) supplied with 16 SMP 3 Microspotting pins (Telechem). Each glass slide contains 48 blocks of 134 spots corresponding to the 128 allele-specific probes, the 5 background controls and the positive control CS05. Similar probes were spotted as distant as possible from each other. DNA was cross-linked by UV irradiation at 250 mJ/cm² (Stratalinker model 1800 UV Illuminator, Stratagene). To prevent non-specific hybridisation, the slides were incubated with 100 μl of prehybridisation solution [400 ng/μl yeast tRNA (Roche), 400 ng/μl herring sperm DNA (Gibco BRL), 5× Denhardt's solution, 3.2×SSC and 0.4% SDS] at 65° C. for 30 min. Prior to hybridisation, the slide containing the prehybridisation mixture was incubated for 2 min at 80° C. to denature the spotted DNA. After prehybridisation, the slides were washed twice in 2×SSC for 5 min at room temperature and dehydrated with a series of 70 (twice), 90 and 100% ethanol, for 5 min each, respectively.

A multiplex PCR for only the six platelet antigens was performed using 5 nM of each HPA and gov primer (7.5 nM of each HPA-3 primer) and 1.2 μM of each Cy5 labeled universal primer. Further, the multiplex PCR was performed as described for the multiplex PCR in Example 2. For hybridisation reaction chambers were made as described by Pastinen et al. (2000). A silicon rubber grid was used to divide the glass slide in 24 different compartments. While the slide was preheated (65° C. for 75 min), 40 μl of the multiplex PCR products was denatured at 95° C. for 5 min, put on ice and 120 μl of ice-cold hybridisation solution [2 ng Cy5-labelled complement of CS05 (5′-catgagctagaagtcaggac-3′) and 4×SSC] was added. For hybridisation, 80 μl of this mixture was added per array and incubated for 3½ hours at 56° C. Next, slides were lifted out of the hybridisation holder in 600 ml 3×SSC and washed twice with 2×SSC/0.1% SDS at 50° C. and twice with 0.2×SSC at RT, for 5 min each, respectively. Glass slides were dried by centrifugation at 800 rpm for 5 min and scanned in an Agilent G2565BA micro array scanner at 10 μm resolution. Photomultiplier tube voltage was always set at 100%. PCR products of six different donors were analysed per slide as shown in FIG. 3. After Genepix analysis the obtained data was converted to Excel. A panel of 12 donor samples with known HPA typing was used to define the HPA scoring criteria. Table III lists the scoring criteria. The median signal intensities F_med of the negative controls (C) were averaged and this background value plus three times its standard deviation was subtracted from the median intensity F_med of each oligo, giving a threshold value, F_th, shown in the third column in Table III. If the F_th showed a negative value for both the matched (allele a) and the mismatched (allele b) probe, the primer pair was excluded for the genotyping result. For genotyping, the ratio of the intensities of two oligonucleotides (one from allele a and the other from allele b) in one set was calculated. The ratios were calculated of the F_med−F_bg intensities for the a-allele to the sum of the F_med−F_bg intensities for both alleles in the sixth column. This formula a/(a+b) has also been described by Hinds et al. (2004). In principle, these ratios should take on values near 1.0, 0.5 or 0.0 for aa, ab or bb genotypes. Only positive F_med−F_bg values were used and therefore negative values were set to 1 in the fifth column. Next, the ratios were converted to genotypes in the seventh column. For HPA-1, -2, -4 and -5 ratio values above 0.75 were typed aa, below 0.25 bb and between 0.35 and 0.65 ab. Due to a high binding of the PCR fragments to the probes representing the a allele, the criteria for HPA-3 and Gov were adjusted to 0.95 and 0.8 for aa, 0.45 and 0.35 for bb and for ab between 0.55 and 0.85 and between 0.45 and 0.7, respectively. The most frequent genotype was coloured orange in the seventh column. This was done for every blood group, every block and every sample. The samples of the 12 donors were used to accept probe-sets that correctly predicted the genotype. As criteria used for the final scoring: 50% of the used probe-sets should predict a genotype if a homozygote genotype was predicted and 40% if a heterozygote genotype was predicted (30% for HPA-3). Using these criteria a blind panel of 58 samples were genotyped. The results of nine of them are shown in Table IV. The genotypes of all 58 samples are listed in Table V. The genotypes of 56 samples could be scored with the preset criteria. Only in one sample a result discrepant with its known genotype was obtained. In this case, 33.3% of the probe sets indicated an HPA-3bb genotype and 37.5% indicated an HPA-3ab genotype as shown in Table VI. Adjustment of the scoring criteria leads to complete concordance of the typing results.

Example 2

A primer mix was constructed with the primers listed in Table II. The concentration of the primers in the PCR mixture is also listed in Table II. The Qiagen multiplex kit was used for the multiplex PCR. In the multiplex PCR, the annealing temperature was not changed during PCR and two fluorescent labelled universal primers were used. In more detail, at the start the PCR mixture contains a very low amount of chimeric primers (5 nM) and an excess of fluorescent-labelled universal primers (0.2 μM of each universal primer per chimeric primer). The temperature profile of the PCR started with 15 min at 95° C., followed by 45 cycles of 94° C. for 30 sec, 57° C. for 90 sec, 72° C. for 90 sec and the protocol ended with a final polymerization step at 72° C. for 10 min. Very little amount of PCR product is amplified during the first amplification cycles, but enough to be used as template by the universal MAPH primers in the following cycles as can be seen on 8% acrylamide gel in FIG. 1. By tagging the MAPH primers at their 5′end with the fluorescent label Cy5, only two fluorescent-labelled primers are necessary to label the PCR product efficiently. As described by White et al. (2002), a better discrmination of the multiplex PCR products can be visualized on an ABI 3700 capillary sequencer (Applied Biossystems), in this case only MAPH-rev is fluorescent (FAM) labeled. Multiplex PCR with all designed PCR primers yielded the expected pattern of peaks as shown in the chromatogram in FIG. 2. This means that all gene-fragments were amplified at similar yields.

TABLE I
Sequences and melting temperatures of the
probes.
Probe	Tm	Sequence (5′-3′)
HPA-1aa	65.86	tacaggccctgcctctggg	+TL,32
HPA-1ab	66.92	aggccctgcctctgggct
HPA-1ac	67.79	ccctgcctctgggctcacc
HPA-1ad	67.35	tgcctctgggctcacctcg
HPA-1ae	64.65	ctctgggctcacctcgctg
HPA-1aa CR	64.65	cagcgaggtgagcccagag
HPA-1ab CR	67.35	cgaggtgagcccagaggca
HPA-1ac CR	67.79	ggtgagcccagaggcaggg
HPA-1ad CR	66.92	agcccagaggcagggcct
HPA-1be CR	65.86	cccagaggcagggcctgta
HPA-1ba	69.93	tacaggccctgcctccggg
HPA-1bb	70.94	aggccctgcctccgggct
HPA-1bc	68.84	ccctgcctccgggctcac
HPA-1bd	66.21	ctgcctccgggctcacct
HPA-1be	66.33	ctccgggctcacctcgct
HPA-1ba CR	66.33	agcgaggtgagcccggag
HPA-1bb CR	66.21	aggtgagcccggaggcag
HPA-1bc CR	68.84	gtgagcccggaggcaggg
HPA-1bd CR	70.94	agcccggaggcagggcct
HPA-1be CR	69.93	cccggaggcagggcctgta
HPA-2aa	63.81	ctgacgcccacacccaag
HPA-2ab	65.21	ctcctgacgcccacaccc
HPA-2ac	65.37	ggctcctgacgcccacac
HPA-2ad	66.12	agggctcctgacgcccac
HPA-2ae	68.84	ccagggctcctgacgccc
HPA-2aa CR	63.81	cttgggtgtgggcgtcag
HPA-2ab CR	65.21	gggtgtgggcgtcaggag
HPA-2ac CR	65.37	gtgtgggcgtcaggagcc
HPA-2ad CR	66.12	gtgggcgtcaggagccct
HPA-2ae CR	68.84	gggcgtcaggagccctgg
HPA-2ba	64.88	cctgatgcccacacccaag
HPA-2bb	65.57	ctcctgatgcccacaccca
HPA-2bc	66.37	ggctcctgatgcccacacc
HPA-2bd	66.45	agggctcctgatgcccaca
HPA-2be	66.45	ccagggctcctgatgccc
HPA-2ba CR	64.88	cttgggtgtgggcatcagg
HPA-2bb CR	65.57	tgggtgtgggcatcaggag
HPA-2bc CR	66.37	ggtgtgggcatcaggagcc
HPA-2bd CR	66.45	tgtgggcatcaggagccct
HPA-2be CR	66.45	gggcatcaggagccctgg
HPA-3aa	69.45	ccatccccagcccctccc
HPA-3ab	69.60	gcccatccccagcccctc
HPA-3ac	70.57	ctgcccatccccagcccc
HPA-3ad1	70.72	gctgcccatccccagccc
HPA-3ad	70.72	ggctgcccatccccagcc
HPA-3ad2	70.72	gggctgcccatccccagc
HPA-3ae	70.08	ggggctgcccatcccca
HPA-3aa CR	69.45	gggaggggctggggatgg
HPA-3ab CR	69.60	gaggggctggggatgggc
HPA-3ac CR	70.57	ggggctggggatgggcag
HPA-3ad1 CR	70.72	gggctggggatgggcagc
HPA-3ad CR	70.72	ggctggggatgggcagcc
HPA-3ad2 CR	70.72	gctggggatgggcagccc
HPA-3ae CR	70.08	tggggatgggcagcccc
HPA-3ba	68.75	ccagccccagcccctcc
HPA-3bb	70.39	gcccagccccagcccct
HPA-3bc	69.91	ctgcccagccccagccc
HPA-3bd1	70.07	gctgcccagccccagcc
HPA-3bd	70.07	ggctgcccagccccagc
HPA-3bd2	69.91	gggctgcccagccccag
HPA-3be	72.50	ggggctgcccagcccca
HPA-3ba CR	68.75	ggaggggctggggctgg
HPA-3bb CR	70.39	aggggctggggctgggc
HPA-3bc CR	69.91	gggctggggctgggcag
HPA-3bd1 CR	70.07	ggctggggctgggcagc
HPA-3bd CR	70.07	gctggggctgggcagcc
HPA-3bd2 CR	69.91	ctggggctgggcagccc
HPA-3be CR	72.50	tggggctgggcagcccc
HPA-4aa	62.77	gccacccagatgcgaaag
HPA-4ab	59.94	cacccagatgcgaaagct
HPA-4ac	61.08	cccagatgcgaaagctca
HPA-4ad	57.99	cagatgcgaaagctcacc
HPA-4ae	57.99	gatgcgaaagctcaccag
HPA-4aa CR	57.99	ctggtgagctttcgcatc
HPA-4ab CR	57.99	ggtgagctttcgcatctg
HPA-4ac CR	61.08	tgagctttcgcatctggg
HPA-4ad CR	59.94	agctttcgcatctgggtg
HPA-4ae CR	62.77	ctttcgcatctgggtggc
HPA-4ba	60.20	gccacccagatgcaaaag
HPA-4bb	61.19	ccacccagatgcaaaagct
HPA-4bc	60.26	acccagatgcaaaagctcac
HPA-4bd	58.50	cagatgcaaaagctcacca
HPA-4be	58.04	gatgcaaaagctcaccagtaa
HPA-4ba CR	58.04	ttactggtgagcttttgcatc
HPA-4bb CR	58.50	tggtgagcttttgcatctg
HPA-4bc CR	60.26	gtgagcttttgcatctgggt
HPA-4bd CR	61.19	agcttttgcatctgggtgg
HPA-4be CR	60.20	cttttgcatctgggtggc
HPA-5aa	57.46	gagtctacctgtttactatcaaagagg
HPA-5ab	56.77	agtctacctgtttactatcaaagaggta
HPA-5ac	57.08	gtctacctgtttactatcaaagaggtaa
HPA-5ad	57.16	ctacctgtttactatcaaagaggtaaaa
HPA-5ae	57.38	acctgtttactatcaaagaggtaaaaa
HPA-5aa CR	57.38	tttttacctctttgatagtaaacaggt
HPA-5ab CR	57.16	ttttacctctttgatagtaaacaggtag
HPA-5ac CR	57.08	ttacctctttgatagtaaacaggtagac
HPA-5ad CR	56.77	tacctctttgatagtaaacaggtagact
HPA-5ae CR	57.46	cctctttgatagtaaacaggtagactc
HPA-5ba	57.42	gagtctacctgtttactatcaaaaagg
HPA-5bb	56.75	agtctacctgtttactatcaaaaaggta
HPA-5bc	57.05	gtctacctgtttactatcaaaaaggtaa
HPA-5ad	57.13	ctacctgtttactatcaaaaaggtaaaa
HPA-5be	57.34	acctgtttactatcaaaaaggtaaaaa
HPA-5ba CR	57.34	tttttacctttttgatagtaaacaggt
HPA-5bb CR	57.13	ttttacctttttgatagtaaacaggtag
HPA-5bc CR	57.05	ttacctttttgatagtaaacaggtagac
HPA-5bd CR	56.75	tacctttttgatagtaaacaggtagact
HPA-5be CR	57.42	cctttttgatagtaaacaggtagactc
Gov-aa	58.44	ttattatcttgacttcagttacaggattt
Gov-ab	59.20	tcttgacttcagttacaggatttacc
Gov-ac	59.15	tgacttcagttacaggatttaccaa
Gov-ad	58.36	cttcagttacaggatttaccaagaat
Gov-ae	59.44	cagttacaggatttaccaagaatttg
Gov-aa CR	59.44	caaattcttggtaaatcctgtaactg
Gov-ab CR	58.36	attcttggtaaatcctgtaactgaag
Gov-ac CR	59.15	tggtaaatcctgtaactgaagtcaa
Gov-ad CR	59.20	ggtaaatcctgtaactgaagtcaaga
Gov-ae CR	58.44	aaatcctgtaactgaagtcaagataataa
Gov-ba	58.35	tatcttgacttcagttccaggatt
Gov-bb	58.39	cttgacttcagttccaggatttac
Gov-bc	59.51	gacttcagttccaggatttacca
Gov-bd	58.24	ttcagttccaggatttaccaag
Gov-be	59.79	cagttccaggatttaccaagaatt
Gov-ba CR	59.79	aattcttggtaaatcctggaactg
Gov-bb CR	58.24	cttggtaaatcctggaactgaa
Gov-bc CR	59.51	ggtaaatcctggaactgaac
Gov-bd CR	58.39	gtaaatcctggaactgaagtcaag
Gov-be CR	58.35	aatcctggaactgaagtcaagata
a33	63.03	tcagccctatcgcaggatgt
a35	53.75	gagacacttgacagtagcca
a38	61.40	ggcagggcacctcagtttat
a42	57.37	tcaccagccagactgtgtag
a43	60.02	cttcacgcaagttgtccaga
CS05	52.41	gtcctgacttctagctcatg
Each oligonucleotide probe has an amine group and a C6 spacer at its
The polymorphic sites in the allele-specific probes are highlighted.

TABLE II
Primers used for multiplex PCR
	Product	concentration
MAPH	Ag		size	of primer
PCR-primer	system	Sequence	Tm*	(bp)	in mpx (nM)
HPA1-left	HPA-1	gccgcgaattcactagtgcttcaggtcacagcgaggt	58.98		5
HPA1-right	HPA-1	ggccgcgggaattcgattgctccaatgtacggggtaaa	59.82	185	5
HPA2-left	HPA-2	gccgcgaattcactagtgtgaaaggcaatgagctgaag	59.15		5
HPA2-right	HPA-2	ggccgcgggaattcgattagccagactgagcttctcca	60.28	113	5
HPA3-left	HPA-3	gccgcgaattcactagtggcctgaccactcctttgc	59.35		7.5
HPA3-right	HPA-3	ggccgcgggaattcgattggaagatctgtctgcgatcc	59.77	141	7.5
HPA4-left	HPA-4	gccgcgaattcactagtgatccgcaggttactggtgag	60.13		5
HPA4-right	HPA-4	ggccgcgggaattcgattccatgaaggatgatctgtgg	58.88	121	5
HPA5-left	HPA-5	gccgcgaattcactagtgtgcaaatgcaagttaaattaccag	59.47		5
HPA5-right	HPA-5	ggccgcgggaattcgattacagacgtgctcttggtaggt	58.88	190	5
Gov-left	Gov	gccgcgaattcactagtgtgtatcagttcttggttttgtgatg	60.31		5
Gov-right	Gov	ggccgcgggaattcgattaaaaccagtagccacccaag	59.10	156	5
JK1/2-left	Kidd	gccgcgaattcactagtggtctttcagccccatttgag	59.67		10
JK1/2-right	Kidd	ggccgcgggaattcgattgttgaaaccccagagtccaa	59.94	86	10
FY1/2-left	Duffy	gccgcgaattcactagtggaattcttcctatggtgtgaatga	59.38		5
FY1/2-right	Duffy	ggccgcgggaattcgattaagaagggcagtgcagagtc	59.60	153	5
GATAbox-left	Duffy	gccgcgaattcactagtgggccctcattagtccttgg	59.49		5
GATAbox-right	Duffy	ggccgcgggaattcgattgaaatgaggggcatagggata	60.12	200	5
KEL1/2-left	Kell	gccgcgaattcactagtgaagggaaatggccatactga	59.39		5
KEL1/2-right	Kell	ggccgcgggaattcgattagctgtgtaagagccgatcc	59.46	207	5
KEL3/4-left	Kell	gccgcgaattcactagtggcctcagaaactggaacagc	60.00		10
KEL3/4-right	Kell	ggccgcgggaattcgattagcaaggtgcaagaacactct	59.15	103	10
RHCEex2for	Rhesus	gccgcgaattcactagtgcgtctgcttccccctcc	61.78		5
RHex2rev	Rhesus	ggccgcgggaattcgattctgaacagtgtgatgaccacc	59.01	268	5
RHCEex5-left	Rhesus	gccgcgaattcactagtgggatgttctggccaagtg	57.47		5
RHex5rev	Rhesus	ggccgcgggaattcgattggctgtcaccacactgactg	60.37	143	5
RHDΨ-left	Rhesus	ggccgcgggaattcgattgtagtgagctggcccatca	59.80		5
RHDΨ-right	Rhesus	gccgcgaattcactagtgtgtctagtttcttaccggcaagt	59.38	380	5
RHD-leftB	Rhesus	gccgcgaattcactagtgttataataacacttgtccacaggg	57.25		5
RHD-rightC	Rhesus	ggccgcgggaattcgattcggctccgacggtatc	58.61	182	5
BigC-left	Rhesus	gccgcgaattcactagtgggccaccaccatttgaa	58.77		15
BigC-rightintron2	Rhesus	ggccgcgggaattcgattccatgaacatgccacttcac	59.97	393	15
MN-left	MNS	gccgcgaattcactagtgtgagggaatttgtcttttgca	60.60		5
MN-right	MNS	ggccgcgggaattcgattcagaggcaagaattcctcca	60.33	394	5
U-left	MNS	gccgcgaattcactagtgcgctgatgttatctgtcttatttttc	59.64		5
U-right	MNS	ggccgcgggaattcgattgatcgttccaataataccagcc	59.71	175	5
JO-left	Dombrock	gccgcgaattcactagtgcctggcttaaccaaggaaaa	59.20		5
JO-right	Dombrock	ggccgcgggaattcgatttcatactgctgtggagtcctg	58.89	181	5
MAPH-reverse#		gccgcgaattcactagtg	57.94		200
MAPH-forward#		ggccgcgggaattcgatt	67.55		200
*Tm values of chimeric primers are without MAPH-tags.
#Cy5 labelled at 5′-end
The gene specific part of the RHDΨ-right and the RHD-rightC primer has been published (Maaskant-van Wijk et al. Transfusion 39, 546, 1999). Published primer sequences Rex6AD3: 5′-TGTCTAGTTTCTTACCGGCAAGT-3′; R1068: 5′-ATTGCCGGCTCCGACGGTATC-3′)
The gene specific part of BigC-left has been published as part of a published primer and part of the bigC-rightintron2, RHex2rev, RHCEex5for primers have been published (Tax et al. Transfusion 42, 634-44, 2002).
Published primer sequences
R348: 5′-CTGAACAGTGTGATGACC-3′;
c/rev: 5′-TGATGACCACCTTCCCAGG-3′;
R637: 5′-GCCCTCTTCTTGTGGATG-3′;
C/for: 5′-CAGCGCCACCACCATTTGAA-3′;
C/rev: 5′-GAACATCCCACTTCACTCCAG-3′)
Part of the MN-primers has been published (Akane et al. Vox Sang, 79, 183-7, 2000)
Published primer sequences MN-Left: 5′-GAGGGAATTTGTCTTTTGCA-3′ and MN-Right: 5′-AGAGGCAAGAATTCCTCC-3′.

TABLE III
Scorings criteria for genotyping HPA-1
ID	Fmed	Fth	Fmed− Fbg	pos(Fmed− Fbg)	ratio	genotype
C a33	861						av. C	692
C a35	579						SD	105
C a38	688						3*SD	315
C a42	640							aa >	0.75
C a43	692							bb <	0.25
CS05	64433							0.35 < ab <	0.65
HPA-1aa	1311	304.2	619	619	0.0	bb
HPA-1aa CR	680	−327	−12	1	0.0	bb
HPA-1ab	911	−95.8	219	219	0.0	bb
HPA-1ab CR	613		−79	1	0.0
HPA-1ac	7100	6093	6408	6408	0.2	bb
HPA-1ac CR	884	−123	192	192	0.2	bb
HPA-1ad	7319	6312	6627	6627	0.2	bb
HPA-1ad CR	614	−393	−78	1	0.0	bb
HPA-1ae	39718	38711	39026	39026	0.4	ab
HPA-1ae CR	792	−215	100	100	0.2	bb
HPA-1ba	29825	28818	29133	29133
HPA-1ba CR	1159	152.2	467	467
HPA-1bb	10446	9439	9754	9754
HPA-1bb CR	827		135	135
HPA-1bc	26518	25511	25826	25826
HPA-1bc CR	1548	541.2	856	856
HPA-1bd	30718	29711	30026	30026
HPA-1bd CR	1276	269.2	584	584
HPA-1be	65375	64368	64683	64683
HPA-1be CR	1060	53.18	368	368
Most frequent genotype results of a sample are in bold. Excluded genotype result bold and boxed.

TABLE IV
Genotyping results of 9 samples of the blind panel
Most frequent genotype results of a sample are in bold. Excluded genotype results are in bold and boxed.

TABLE V

Genotypes of blind panel of 58 samples

HPA-

% pos

% pos

% pos

% pos

% pos

Sample

1

HPA-1

HPA-2

HPA-2

HPA-3

HPA-3

HPA-4

HPA-4

HPA-5

HPA-5

Gov

% pos gov

1

aa

100%

28/28

aa

94%

30/32

bb

54%

13/24

aa

85%

17/20

aa

75%

15/20

ab

75%

27/36

2

aa

96%

24/25

aa

100%

32/32

aa

83%

20/24

aa

100%

20/20

aa

79%

15/19

ab

64%

23/36

3

bb

70%

19/27

aa

100%

31/31

ab

50%

12/24

aa

84%

16/19

ab

80%

16/20

bb

72%

26/36

4

ab

43%

10/23

aa

100%

32/32

aa

79%

19/24

aa

94%

17/18

ab

45%

5/11

aa

67%

24/36

5

aa

96%

27/28

aa

100%

32/32

aa

83%

20/24

aa

100%

20/20

aa

68%

13/19

aa

94%

34/36

6

aa

93%

25/27

ab

69%

22/32

ab

42%

10/24

aa

100%

20/20

ab

53%

10/19

aa

58%

21/36

7

ab

54%

15/28

ab

81%

26/32

aa

79%

19/24

aa

100%

20/20

ab

60%

12/20

ab

72%

26/36

8

aa

96%

27/28

aa

100%

32/32

aa

83%

20/24

aa

95%

19/20

bb

65%

13/20

ab

81%

29/36

9

bb

81%

21/26

aa

100%

32/32

ab

38%

9/24

aa

90%

18/20

ab

65%

13/20

ab

78%

28/36

10

aa

96%

27/28

ab

72%

23/32

aa

79%

19/24

aa

95%

19/20

aa

85%

17/20

ab

83%

30/36

11

bb

70%

16/23

aa

97%

30/31

bb

44%

7/16

aa

93%

13/14

bb

81%

13/16

aa

83%

30/36

12

aa

100%

23/23

ab

55%

17/31

ab

46%

11/24

aa

83%

10/12

aa

80%

16/20

bb

75%

27/36

13

ab

54%

15/28

ab

63%

20/32

ab

50%

12/24

aa

85%

17/20

ab

65%

13/20

aa

64%

23/36

14

ab

44%

12/27

bb

94%

30/32

ab

50%

12/24

aa

100%

20/20

aa

85%

17/20

bb

64%

23/36

15

aa

93%

26/28

aa

100%

32/32

ab

38%

9/24

aa

90%

18/20

ab

55%

11/20

aa

72%

26/36

16

aa

96%

27/28

aa

75%

24/32

ab

42%

10/24

aa

89%

17/19

ab

70%

14/20

ab

81%

29/36

17

bb

74%

20/27

ab

53%

17/32

ab

39%

9/23

aa

95%

19/20

aa

78%

14/18

ab

67%

24/36

18

aa

93%

25/27

ab

81%

26/32

ab

33%

8/24

aa

75%

15/20

ab

75%

15/20

aa

94%

34/36

19

aa

96%

27/28

ab

78%

25/32

ab

54%

13/24

aa

100%

20/20

aa

65%

13/20

ab

69%

25/36

20

bb

75%

21/28

aa

97%

31/32

aa

83%

20/24

aa

100%

20/20

aa

90%

18/20

ab

64%

23/36

21

aa

100%

28/28

ab

66%

21/32

bb

61%

14/23

aa

95%

19/20

aa

100%

20/20

bb

61%

22/36

22

aa

96%

26/27

aa

100%

32/32

aa

88%

21/24

aa

90%

18/20

aa

80%

16/20

bb

83%

30/36

23

aa

100%

28/28

aa

100%

29/29

aa

88%

21/24

aa

85%

17/20

aa

80%

16/20

bb

78%

28/36

24

aa

100%

28/28

aa

100%

31/31

ab

42%

10/24

aa

100%

20/20

ab

65%

13/20

bb

53%

19/36

25

bb

85%

22/26

ab

78%

25/32

ab

38%

9/24

aa

72%

13/18

aa

79%

15/19

aa

94%

34/36

26

aa

96%

27/28

aa

100%

32/32

aa

79%

19/24

aa

95%

18/19

ab

95%

18/19

ab

64%

23/36

27

aa

81%

21/26

aa

100%

31/31

aa

63%

15/24

aa

86%

12/14

aa

100%

19/19

ab

64%

23/36

28

aa

79%

22/28

aa

100%

32/32

bb

68%

16/22

aa

80%

16/20

aa

75%

15/20

ab

72%

26/36

29

aa

100%

28/28

ab

69%

22/32

ab

54%

13/24

aa

84%

16/19

aa

80%

16/20

ab

67%

24/36

30

bb

70%

16/23

aa

100%

32/32

bb

72%

13/18

aa

85%

11/13

aa

94%

15/16

ab

54%

19/35

31

bb

74%

20/27

bb

97%

31/32

ab

50%

12/24

aa

90%

18/20

aa

90%

18/20

bb

58%

21/36

32

aa

89%

25/28

aa

100%

32/32

bb

52%

12/23

aa

100%

20/20

aa

65%

13/20

aa

86%

31/36

33

aa

96%

24/25

aa

94%

30/32

aa

63%

15/24

aa

94%

17/18

ab

60%

12/20

ab

64%

23/36

34

bb

75%

18/24

aa

100%

32/32

aa

71%

17/24

aa

100%

19/19

ab

76%

13/17

ab

78%

28/36

35

aa

93%

25/27

ab

41%

13/32

ab

46%

11/24

aa

84%

16/19

aa

68%

13/19

ab

69%

25/36

36

ab

48%

13/27

aa

100%

32/32

bb

52%

11/21

aa

85%

17/20

aa

100%

20/20

aa

86%

31/36

37

aa

100%

28/28

ab

72%

23/32

bb

63%

15/24

aa

100%

19/19

aa

100%

20/20

ab

78%

28/36

38

aa

96%

27/28

ab

78%

25/32

ab

46%

11/24

aa

100%

20/20

aa

85%

17/20

ab

78%

28/36

39

ab

48%

13/27

ab

91%

29/32

ab

50%

12/24

aa

80%

16/20

aa

80%

16/20

ab

75%

27/36

40

ab

44%

12/27

aa

100%

32/32

bb

64%

14/22

aa

84%

16/19

ab

65%

13/20

aa

86%

31/36

41

ab

56%

15/27

aa

100%

32/32

ab

54%

13/24

aa

100%

19/19

aa

65%

11/17

ab

75%

27/36

42

aa

100%

28/28

ab

59%

19/32

ab

54%

13/24

aa

90%

18/20

aa

100%

20/20

bb

67%

24/36

43

aa

96%

27/28

ab

72%

23/32

aa

79%

19/24

aa

100%

20/20

aa

100%

20/20

bb

53%

19/36

44

aa

100%

28/28

aa

100%

32/32

bb

71%

17/24

aa

95%

19/20

aa

90%

18/20

bb

53%

19/36

45

aa

100%

28/28

ab

63%

20/32

bb

68%

15/22

aa

100%

20/20

aa

95%

19/20

bb

56%

20/36

46

ab

35%

7/20

aa

94%

30/32

ab

35%

8/23

aa

79%

11/14

aa

83%

15/18

ab

61%

22/36

47

aa

81%

21/26

ab

78%

25/32

ab

50%

12/24

aa

71%

12/17

aa

73%

11/15

ab

69%

24/35

48

aa

93%

25/27

ab

94%

30/32

ab

42%

10/24

aa

84%

16/19

aa

80%

16/20

ab

64%

23/36

49

bb

70%

19/27

aa

100%

32/32

ab

67%

16/24

aa

100%

20/20

aa

63%

10/16

bb

64%

23/36

50

aa

93%

26/28

aa

100%

32/32

ab

46%

11/24

aa

95%

19/20

aa

80%

16/20

ab

72%

26/36

51

aa

96%

27/28

ab

72%

23/32

aa

83%

20/24

aa

85%

17/20

aa

75%

15/20

ab

61%

22/36

52

aa

100%

22/22

aa

100%

32/32

aa

79%

19/24

aa

89%

16/18

aa

84%

16/19

bb

75%

27/36

53

bb

71%

17/24

aa

100%

32/32

bb

64%

14/22

aa

94%

15/16

aa

78%

14/18

bb

69%

25/36

54

aa

89%

24/27

aa

100%

32/32

ab

46%

11/24

aa

83%

15/18

ab

67%

12/18

ab

67%

24/36

55

aa

100%

27/27

aa

100%

32/32

ab

50%

12/24

aa

100%

19/19

aa

90%

18/20

aa

83%

30/36

56

bb

76%

19/25

aa

100%

32/32

aa

75%

18/24

aa

100%

20/20

bb

63%

12/19

bb

78%

28/36

57

aa

100%

27/27

aa

100%

32/32

bb

80%

16/20

aa

100%

20/20

bb

68%

13/19

ab

72%

26/36

58

ab

50%

14/28

bb

100%

32/32

ab

50%

12/24

aa

70%

14/20

aa

75%

15/20

bb

83%

30/36

Genotypes are in bold when the predicted genotype is higher then 50% for the homozygote genotypes or 40% for the heterozygote genotypes (HPA-3ab >30%)

TABLE VI
HPA-3 genotyping results of sample 9 compared with the results of 6 other samples
Most frequent genotype results of a sample are in bold. Excluded genotypes are in bold and boxed. Genotype of sample 9 should be HPA-3bb and therefore the bb-genotypes are shown in capital letters for this sample.

TABLE VII
Additionally tested primers for multiplex PCR
These primers have been tested together with primers of SEQ ID NOS:15 to 40 in
one multiplex PCR. PCR products of the correct product size were obtained.
	Product
Ag		size
system	MAPH PCR-primer	Sequence	Tm*	(bp)
Duffy	Fyx-left (MAPH)	gccgcgaattcactagtgTCATGCTTTTCAGACCTCTCTTC	60.01
Duffy	Fyx-right (MAPH2)	ggccgcgggaattcgattCAAGACGGGCACCACAAT	60.53	143
Kell	KEL6/7-left (MAPH)	gccgcgaattcactagtgGCAGCACCAACCCTATGTTC	60.53
Kell	KEL6/7-right (MAPH)	ggccgcgggaattcgattTCAGGCACAGGTGAGCTTC	60.13	154
Rhesus	RHDex3-left (MAPH)	gccgcgaattcactagtgTCCTGGCTCTCCCTCTCT	57.48
Rhesus	RHCEex3-right (MAPH)	ggccgcgggaattcgattTTTTTCAAAACCCCGGAAG	59.90	294
Rhesus	RhDVI-left (MAPH) (fw)	ggccgcgggaattcgattCTTTGAATTAAGCACTTCACAGA	56.48
Rhesus	RhDVI-right (MAPH) (rev)	gccgcgaattcactagtgGCCAGAATCACACTCCTGCT	60.42	277
MNS	Ss-left (MAPH3)	gccgcgaattcactagtgTTTTTCTTTGCACATGTCTTT	55.20
MNS	Ss-right (MAPH2)	ggccgcgggaattcgattTCTTTGTCTTTACAATTTCGTGTG	58.42	276
Dombrock	DO-left (MAPH)	ggccgcgggaattcgattTGATCCCTCCCTATGAGCTG	60.17
Dombrock	DO-right (MAPH)	gccgcgaattcactagtgTTATATGTGCTCAGGTTCCCAGT	59.91	136
Colton	Colton-left (MAPH)	gccgcgaattcactagtgGCCACGACCCTCTTTGTCT	60.25
Colton	Colton-right (MAPH)	ggccgcgggaattcgattTACATGAGGGCACGGAAGAT	60.48	269
Diego	Diego-left (MAPH)	gccgcgaattcactagtgACTTATTCACGGGCATCCAG	59.96
Diego	Diego-right (MAPH)	ggccgcgggaattcgattAAGCTCCACGTTCCTGAAGA	59.99	185
Diego	Wr-left (MAPH)	gccgcgaattcactagtgGGCTTCAAGGTGTCCAACTC	59.70
Diego	Wr-right (MAPH)	ggccgcgggaattcgattAGGATGAAGACCAGCAGAGC	59.56	158
Yt	Yt-left (MAPH)	ggccgcgggaattcgattCCTTCGTGCCTGTGGTAGAT	60.13
Yt	Yt-right (MAPH)	gccgcgaattcactagtgTTCTGGGACTTCTGGGAATG	60.04	235
Lutheran	Lu-left (MAPH)	gccgcgaattcactagtgGGACCCAGAGAGAGAGAGACTG	59.61
Lutheran	Lu-right (MAPH)	ggccgcgggaattcgattGGGAGTCCAGCTGGTATGG	60.48	220
*Tm values are without MAPH tags.

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Claims

1. A method to amplify and detectable label a region of the loci of at least two different blood cell antigens that contain a site of a nucleotide polymorphism of said blood cell antigen which method comprises subjecting DNA from an individual of a mammalian species to a multiplex Polymerase Chain Reaction (PCR), wherein said each PCR of said multiplex employs at least one pair of chimeric blood cell antigen-specific primers for each blood cell antigen to be genotyped and at least one detectably labeled universal primer, wherein said at least one universal primer has a unique sequence not occurring in the DNA of said mammalian species, and wherein each chimeric primer pair comprises a left chimeric primer and a right chimeric primer, each comprising a blood cell antigen-specific part at the 3′ end and a universal part at the 5′ end, wherein the nucleotide sequence of the universal part of the chimeric primers corresponds to the nucleotide sequence of said at least one universal primer, and wherein said blood cell antigen-specific parts of the chimeric primer pair enclose a region of the locus of the blood cell antigen which contains the site of a nucleotide polymorphism of said blood cell antigen.

2. The method of claim 1, wherein said at least one detectably labeled universal primer is used in a molar amount which is at least 100 times the molar amount of each chimeric primer.

3. The method of claim 1, wherein said each PCR employs a pair of detectably labeled universal primers each with a unique sequence not occurring in the DNA of said mammalian species, and wherein for each chimeric primer pair the nucleotide sequence of the universal part of one member of the chimeric primer pair corresponds to the nucleotide sequence of one member of the universal primer pair and the nucleotide sequence of the universal part of the other member of the chimeric primer pair corresponds to the nucleotide sequence of the other member of the universal primer pair.

4. The method of claim 3, wherein one of said universal primers has the base sequence gcegcgaattcactagtg (SEQ ID NO:2) and the other universal primer has the base sequence ggccgcgggaattcgatt (SEQ ID NO:1).

5. The method of claim 1, wherein each universal primer carries a fluorescent label at its 5′ end.

6. The method of claim 1, wherein said mammalian species is human, said DNA is genomic DNA, and said blood cell antigens comprise at least two members selected from the group consisting of HPA1, HPA2, HPA3, HPA4, HPA5, Gov, JK1/2, FY1/2, GATAbox, KEL1/2, KEL3/4, RHCEex2/RHex2, RHCEex5/RHex5, RHDy, RHD, BigC, MN, U and JO.

7. The method of claim 6, wherein said chimeric primer pairs comprise at least two, pairs selected from the group consisting of:

8. The method of claim 1, wherein the DNA polymerase used in the multiplex PCR is a heat-resistant polymerase and each cycle of the multiplex PCR comprises a heat denaturation step of 15 to 60 sec at a temperature of 90 to 98° C., an annealing step of 60 to 120 sec at a temperature of 54 to 60° C., and a primer extension step of 60 to 120 sec at a temperature of 68 to 76° C.

9. The method of claim 1, wherein said multiplex PCR uses, based on a reaction volume of 50 μl , about 100 ng of genomic DNA from said individual, about 5 nM of each chimeric primer, about 0.2 μl of each detectably labeled universal primer, about 25 μl of 2× MasterMix containing buffer, dNTP's and DNA polymerase.

10. The method of claim 1, which further comprises determining the genotype for each of said blood cell antigens by hybridizing the products of the multiplex PCR amplification, after denaturation, to blood cell antigen allele-specific oligonucleotide probes, and analyzing the hybridization pattern.

11. The method of claim 10, wherein the allele-specific probes have a length of 15 to 40 nucleotides.

12. The method of claim 10, wherein the probes comprise for each allele of a blood cell antigen at least two different sense probes and at least two different antisense probes, each of them covering the site of the nucleotide polymorphism but at varying positions.

13. The method of claim 10, wherein probes comprise at least 72 different probes selected from the group consisting of:

14. The method of claim 10, wherein the oligonucleotide probes comprise at their 5′ end a linker and a reactive group for attachment to an array support.

15. The method of claim 10, wherein the probes include one or more oligonucleotides with a sequence not occurring in the DNA of said mammalian species to allow for background subtraction.

16. The method of claim 10, wherein the probes include one or more positive hybridization controls, and its detectably labeled complement is added to the products of the multiplex PCR amplification.

17. The method of claim 10, wherein the probes, before adding the products of the multiplex PCR amplification, are subjected to prehybridization and DNA denaturation treatments.

18. The method of claim 10, wherein the denatured products of the multiplex PCR amplification are applied to the probes without prepurification.

19. The method of claim 10, wherein for each blood cell antigen the ratio of the signal intensities for each of the alleles is used to assign the genotype.

20. A kit for genotyping blood cell antigens by the method of claim 1, comprising one pair of blood cell antigen-specific chimeric primers for each blood cell antigen to be genotyped and at least one detectably labeled universal primer.

21. The kit of claim 20, further comprising a DNA array of probes.

22. A set of blood cell antigen-specific chimeric primer pairs useful in a multiplex PCR, comprising at least two primer pairs selected from the group as defined in claim 7.

23. A set of blood cell antigen allele-specific oligonucleotide probes useful for genotyping blood cell antigens, comprising at least 72 different probes selected from the group as defined in claim 13.

24. The method of claim 10, wherein said probes are contained in a supported array.

25. The method of claim 15, wherein said probes are selected from the group consisting of

26. The method of claim 16, wherein said positive hybridization control is