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A method of genotyping blood cell antigens and kit suitable for genotyping blood cell antigens

An antigen gene, blood cell technology, applied in the direction of biochemical equipment and methods, microbial determination/inspection, etc., can solve the problem that the feasibility of DNA microarrays, the characteristics of oligonucleotide probes and microarray formats are not disclosed, Issues such as practical, rapid and reliable methods for analyzing blood cell antigen genotyping are not published

Inactive Publication Date: 2007-08-29
桑昆血液供给基金会
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0016] To date, DNA microarray methods have not been used for genotyping blood cell antigens, and the prior art discloses neither the feasibility of using DNA microarrays in methods for genotyping large numbers of blood cell antigens nor the suitability for such methods. Characteristics of oligonucleotide probes and microarray formats for genotyping blood cell antigens, nor disclosed practical, rapid and reliable methods for analyzing hybridization results for clinically relevant genotyping of blood cell antigens

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  • A method of genotyping blood cell antigens and kit suitable for genotyping blood cell antigens
  • A method of genotyping blood cell antigens and kit suitable for genotyping blood cell antigens
  • A method of genotyping blood cell antigens and kit suitable for genotyping blood cell antigens

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Embodiment 1

[0796] Table 1 shows the sequences and Tm values ​​of the probes spotted on polylysine-coated slides. The SNPs of HPA-3 and 5 were located in GC-rich and GC-poor regions, respectively. Therefore, these probes are shorter or longer than other oligonucleotides and have Tm values ​​outside 60-65°C. Omnigrid 100 with 16SMP 3 micro-spotting needle (Telechem) was used _ Microarray instrument (Genemachines), will be dissolved in 0.4M NaHCO 3 Probes were spotted at a concentration of 50 [mu]M in (pH 9.4) on poly-L-lysine-coated slides. Each slide contained 48 fields of 134 spots each, corresponding to 128 allele-specific probes, 5 background controls, and positive control CS05. Similar probes are spotted on spots as far apart as possible from each other. by 250mJ / cm 2 (Stratalinkermodel 1800 UV Illuminator, Stratagene) UV irradiation was performed to cross-link DNA. To prevent non-specific hybridization, slides were mixed with 100 μl of prehybridization solution [400ng / μl yeast ...

Embodiment 2

[0799]Primer mixes were constructed with the primers listed in Table 2. Primer concentrations in the PCR mix are also listed in Table 2. Multiplex PCR was performed using the Qiagen multiplex kit. In multiplex PCR, the annealing temperature was not changed during the PCR, and two fluorescently labeled universal primers were used. More specifically, at the beginning, the PCR mix contained a very small amount of chimeric primers (5 nM) and an excess of fluorescently labeled universal primers (0.2 μM of each universal primer per chimeric primer). In terms of temperature, PCR started at 95°C for 15 min, followed by 45 cycles of 94°C for 30 s, 57°C for 90 s, 72°C for 90 s, and the protocol ended with a final polymerization step at 72°C for 10 min. In the first few cycles, very little PCR product was amplified, but as seen by 8% acrylamide gel electrophoresis in Figure 1, it was sufficient to serve as a template for the universal MAPH primer in subsequent cycles. By labeling the ...

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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 lea st 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 k it for genotyping blood cell antigens by this method. A set of blood cell antig en- specific chimeric primer pairs and a set of blood cell antigen allele-specif ic oligonucleotide probes.

Description

technical field [0001] The present invention relates to the field of genotyping blood cell antigens, and more particularly to genotyping antigens on red blood cells (blood group antigens), platelets (platelet antigens) and white blood cells (leukocyte antigens). [0002] The invention provides a method for genotyping blood cell antigens and a kit for genotyping blood cell antigens, as well as a primer set and a probe set for genotyping blood cell antigens. Background technique [0003] When blood or blood components, such as red blood cells, platelets, and white blood cells, are administered from a donor to another human being (or, more broadly, to another mammalian individual), if the donor blood or blood components are not identical to the recipient blood If they match exactly, serious adverse reactions may occur. Well known are transfusion reactions (blood agglutination), which occur, for example, when blood from a donor of blood type A is given to a person of blood type...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/56
CPCC12Q1/686C12Q1/6827
Inventor 西格里德·赫尔马·维尔马·贝布尔亨德里卡·维林加-杰尔斯马约翰内斯·特奥多鲁斯·登邓内恩马申卡·德哈斯
Owner 桑昆血液供给基金会
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