首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 10 毫秒
1.
We have established a precise, rapid, simple and economical subtyping method for alleles encoding the HLA-A2 and -B40 antigens using microtiter plate-reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse dot blot hybridization. Amino-modified sequence-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform high-resolution subtyping of the HLA-A2 and -B40 antigen groups, the alpha1 and alpha2 domain regions were amplified using a pair of group-specific primers composed of an unlabeled sense primer and a biotinylated antisense primer. PCR-amplified products were hybridized with SSO probes in hybridization buffer containing formamide for 1 hour at 37 degrees C. After washing with 2 X SSC at room temperature, the bound PCR products were detected by alkaline phosphatase-conjugated streptavidine followed by color development. All of 8 HLA-B40 suballeles, all of 2 HLA-B47 suballeles (B40 group-specific primers used in this study allowed also B47 amplification) and 17 out of 21 HLA-A2 suballeles were discriminated. The remaining four HLA-A2 suballeles were determined by analysis after exon 4 amplification. HLA-DNA typing by this method was easily and exactly performed regardless of sample number. The greatest advantages of this technique are strong positive signals obtained, reproducibility and the ease of thermoregulation for hybridization and washing as compared to previously reported microtiter plate hybridization methods.  相似文献   

2.
We have established a precise, rapid, simple and practical HLA class I DNA typing method using the microtitre plate–reverse hybridization assay (MRHA), which enables us to perform simultaneous DNA typing of the HLA‐A, ‐B and ‐C loci using the same PCR parameters and hybridization conditions. PCR‐amplified products for the HLA‐A, ‐B and ‐C loci were hybridized, respectively, with sequence‐specific oligonucleotide (SSO) probes, which were immobilized covalently onto a microtitre plate, in hybridization buffer containing formamide at 37 °C. After washing at room temperature, the bound PCR products were detected by peroxidase‐conjugate streptavidine followed by colour development such as enzyme immunoassay (EIA). In addition to the simple thermoregulation for hybridization and postwashing, strong positive signals, low background and high reproducibility, this DNA typing method enabled simultaneous typing of the HLA‐A, ‐B and ‐C loci using a single microtitre plate as in HLA serotyping. The assignment of the HLA genotype was easily achieved by automated colorimetric reading and computer software, based on the cut‐off value (threshold) established for each probe. For routine HLA class I typing, it may be possible to replace serological typing with the HLA class I DNA typing system using our MRHA method.  相似文献   

3.
DNA typing for HLA class II improves the typing quality and this was shown previously to be relevant for kidney graft survival. In this project we addressed the question whether molecular typing for HLA class I also increases the efficacy of HLA matching in kidney transplantation. 215 HLA-A,-B,-DR zero-mismatched donor/recipient pairs as defined by serological typing were selected. Retrospective HLA-A and HLA-B typing was performed both by the PCR-SSP and the PCR-SSOP method. DNA typing for HLA-A revealed discrepant results to serology in 5.7% of the donors and 2.8% of the recipients. HLA-B typing discrepancies were found in 6.6% of the donors and 5.6% of the recipients. 10.4% of the donors and 6.5% of the recipients showed either an HLA-A or an HLA-B discrepancy. Nearly one-third of the HLA-A discrepancies affected A19 splits. The most common reason for HLA-A discrepancies was the erroneous assignment of serological blanks, whereas HLA-B errors were caused mainly by the assignment of incorrect specificities. DNA typing allowed the definition of HLA-A and -B split specificities in all 118 "splitable" cases for which only broad specificities were reported based on serological typing. A total of 183 DNA class I compatible transplants had a 15% higher one-year graft survival rate than 32 transplants for which DNA typing revealed a class I incompatibility.  相似文献   

4.
We describe a rapid, reliable and cost-effective method for intermediate-to-high-resolution sequence-based HLA class I typing using frozen plasma as a source of genomic DNA. The plasma samples investigated had a median age of 8.5 years. Total nucleic acids were isolated from matched frozen PBMC (~2.5 million) and plasma (500 μl) samples from a panel of 25 individuals using commercial silica-based kits. Extractions yielded median [IQR] nucleic acid concentrations of 85.7 [47.0-130.0]ng/μl and 2.2 [1.7-2.6]ng/μl from PBMC and plasma, respectively. Following extraction, ~1000 base pair regions spanning exons 2 and 3 of HLA-A, -B and -C were amplified independently via nested PCR using universal, locus-specific primers and sequenced directly. Chromatogram analysis was performed using commercial DNA sequence analysis software and allele interpretation was performed using a free web-based tool. HLA-A, -B and -C amplification rates were 100% and chromatograms were of uniformly high quality with clearly distinguishable mixed bases regardless of DNA source. Concordance between PBMC and plasma-derived HLA types was 100% at the allele and protein levels. At the nucleotide level, a single partially discordant base (resulting from a failure to call both peaks in a mixed base) was observed out of >46,975 bases sequenced (>99.9% concordance). This protocol has previously been used to perform HLA class I typing from a variety of genomic DNA sources including PBMC, whole blood, granulocyte pellets and serum, from specimens up to 30 years old. This method provides comparable specificity to conventional sequence-based approaches and could be applied in situations where cell samples are unavailable or DNA quantities are limiting.  相似文献   

5.
6.
A group of HLA-A locus alleles known to be comprised of approximately 14 closely related variants are collectively called HLA-A2 and -A28. Variations among these alleles are given by differences in only a few codons, and in the case of A*6901, elements of A*6801 (exons 1 and 2) and of A*0201 are combined. The purpose of these experiments was to determine the possibility of designing oligonucleotide probes to identify and develop a typing method for all or most of the A2 and A28 variants. Because the regions of interest are also shared by alleles of other groups, allele-specific or group-specific primers were needed to amplify only the alleles under study. HLA-A2-specific amplification of exon 2 and selective amplification of portions of exon 3 of the A2-A28 group were accomplished with sequence-specific primers and after appropriate adjustments of the PCR conditions. Hybridization patterns using products of four PCR reactions with our set of probes distinguished 11 alleles. Two other alleles might be recognized with the reagents used, but were not found in the panels in this study. A*0201 and A*0209, which are different in exon 4, were not resolved because exon 4 was not tested. A new variant of Aw68, defined by a hybridization pattern obtained with our probes, was different from A*6801 only in that it was negative with probe A6. It was called A*68.3. Population studies were performed in North American whites, blacks, and Indians and in a sample of subjects from North China. HLA-A*0201 was the most frequent allele. A*0202 was found only in blacks, and A*0203 and A*0207 were found only in Chinese. Among the A28-positive subjects, Caucasoids were predominantly A*6801 or A*68.3; A*6802 was the most frequent subtype in American blacks; among American Indians the predominant type was A*68.3. The two A28-positive Chinese subjects studied had A*6901. The results obtained demonstrate that DNA typing is an efficient method for determining these alleles. The methodology should be applicable to other class I groups and should be useful for more extensive population studies, for matching for bone marrow transplantation, and for investigation of certain diseases associated with HLA class I alleles.  相似文献   

7.
HLA class I and class II DNA typing and the origin of Basques   总被引:3,自引:0,他引:3  
Abstract: Seven HLA class I and class II loci (HLA-A, B, C, DRB1, DQA1, DPA1 and DPB1) were typed at the DNA level in two populations of the Iberian Peninsula (100 Basque and 88 Catalan individuals) in order to unravel their genetic relationship and to compare these results with other European and Mediterranean populations. For the first time,- the frequencies of alleles and haplotypes for the class I HLA loci at the DNA level in these populations are presented. The most frequent haplotype in both populations is A*29-Cw*1601-B*44-DRB1*0701-DQA1*0201-DPA1*0103-DPB1*0401. Neither population differed markedly from the highly homogeneous European and Mediterranean genetic landscape. The Basques, a European outlier population according to classical genetic markers, appear to lie within the genetic European variation with a slight uniqueness and show no clear relationship to North African populations, as has been postulated in some previous HLA studies. Here, the range of possibilities provided by the highly polymorphic HLA system is stressed by using genetic distances, phylogenetic trees and principal component analyses in order to reconstruct population history.  相似文献   

8.
9.
10.
The analysis of cattle MHC (BoLA) class I gene expression is an essential component of studies on immune responses and susceptibility to disease. International BoLA workshops have generated data and reagents that allow discrimination of class I molecules at the haplotype level, but progress has been limited by difficulties encountered in defining single alleles. Our aim in this study was to develop a DNA-based system for improved identification of expressed class I alleles, utilizing available cDNA sequences derived from cattle carrying a series of serologically defined class I specificities. This method has allowed more accurate typing of animals for expression of the class I genes present within a small number of haplotypes. The method has also reliably differentiated between allelic variants (identified by prior sequence analysis) and has split existing serological specificities. The data show that MHC class I genes in cattle are more polymorphic than demonstrated by serology and biochemical analysis.  相似文献   

11.
Abstract: Variable amounts of non-specific amplification may occur in HLA PCR-SSP typing, and this can be significantly reduced by the use of AmpliTaq Gold. In an effort to achieve an optimal balance between specificity and efficiency of the PCR amplification for both HLA alleles and the internal control, we designed a system of timed-release activity by combining two different Taq DNA polymerases. The reaction was started at a relatively low level of enzyme activity and as thermal cycling progressed more and more Ampli-Taq Gold was slowly activated for the reaction to continue. We applied this system to all routine HLA PCR-SSP typing. The number of repeat typings due to non-specific amplification and/or amplification failure of the internal control was remarkably reduced.  相似文献   

12.
Saito S  Ota S  Yamada E  Inoko H  Ota M 《Tissue antigens》2000,56(6):522-529
HLA class I and class II allelic genotypes were determined in 371 unrelated individuals and 309 members of 81 families inhabiting the central Japan area. A total of 20 HLA-A alleles, 16 HLA-Cw alleles, 38 HLA-B alleles, 27 HLA-DRB1 alleles, 15 HLA-DQB1 alleles and 12 HLA-DPB1 alleles were detected. By the two-, three-, four-, five- and six-locus allelic association analyses extracted from the HLA-A to -DPB1 locus, 26 HLA-Cw-B haplotypes, 25 HLA-DRB1-DQB1 haplotypes, 42 HLA-Cw-B-DRB1 haplotypes, 37 HLA-Cw-B-DRB1-DQB1 haplotypes, 29 HLA-A-Cw-B-DRB1-DQB1 haplotypes and 21 HLA-A-Cw-B-DRB1-DQB1-DPB1 haplotypes with the frequencies of higher than 0.005 were recognized. Among 19 HLA-B alleles with the high allele frequencies (above 0.007), 9 HLA-B alleles, B*0702, B*1301, B*3701, B*3901, B*4006, B*4403, B*5201, B*5901 and B*6701 were found to be tightly associated with single HLA-Cw alleles. Most of HLA-DRB1 alleles showed strong associations with single HLA-DQB1 alleles, but DRB1*0802 and DRB1*1401 were associated with two different DQB1 alleles. Extended haplotypes carrying infrequent class I alleles with the allele frequencies of lower than 0.007 were defined by family studies. Gene frequencies and haplotypic associations within the entire HLA classical loci elucidated at the high resolution (four-digital) allelic level will provide useful information on anthropology, marrow donor registry, legal medicine and disease-association studies.  相似文献   

13.
The HLA region on the short arm of chromosome 6 (6p21.3) contains the most polymorphic coding sequences in the human genome. High-resolution DNA-based HLA typing of population samples of the polymorphic class I loci, HLA-A, -B, and -C has only recently become feasible. Here, we report molecular HLA typing on family-based samples of European origin (the CEPH repository), which demonstrated very high polymorphism, with 20 A alleles, 38 B alleles and 19 C alleles in the sample of 248 independent haplotypes. In general, allele frequency distributions are consistently more even (lower observed homozygosity statistic) than expected from a past of selective neutrality suggesting a history of balancing selection. This was also true for the class II loci, DRB1, DQA1 and DQB1 in these samples, but not for the DPA1 and DPB1 loci, whose allelic frequency distributions were more skewed (higher observed homozygosity statistic) than expected under a neutral model. Although linkage disequilibrium is a prominent feature across the HLA region, only 19% of the eight locus haplotypes were sampled more than once. The relative age of some of the B alleles could be inferred from the pattern of B-C haplotypic associations. We suggest that the observed patterns of linkage disequilibrium reflect the operation of selection on nearly all HLA alleles.  相似文献   

14.
Gastric cancer is multifactorial disease and several reports have described genetic factors involved in pathogenesis of gastric cancer. Recently, it was reported that HLA class II gene DQB1*0301 was strongly associated with gastric cancer in Caucasian population (20). We performed DNA typing of HLA class I (HLA-A) and HLA class II genes (HLA-DR, DQ and DP) to elucidate the HLA alleles or HLA haplotypes associated with gastric cancer in Japanese population using polymerase chain reaction-sequence-specific oligonucleotide probe analysis in 88 unrelated patients with gastric cancer and 525 unrelated healthy controls. We observed slight difference in frequencies of some HLA alleles and haplotypes between gastric cancer patients and controls; however, after Bonferroni correction, statistical significance was not confirmed. It is possible that environmental factors such as diet cover the contribution of genetic factors to the disease in Japanese population, which has a higher frequency of gastric cancer than do Caucasian populations, most likely due to more exposure to environmental risk factors.  相似文献   

15.
Abstract: We have identified locus-specific sequences in the first and third introns flanking the polymorphic second and third exons of HLA class I genes. PCR primers derived from these conserved sequences produced DNA fragments of the expected sizes for each of the HLA-A, -B, and -C loci in the amplification of genomic DNA. PCR products generated using each of the locus-specific sets of primers displayed exquisite locus specificity, as assessed by hybridization with oligonucleotide probes specific for ten classical and non-classical HLA class I genes. Amplification with these primer sets was effective and specific for the HLA alleles tested under the given PCR conditions. When hybridized with oligonucleotides derived from shared polymorphic sequence motifs, reaction patterns of PCR products from each locus were precisely as expected from published or database sequences. Chemiluminescent signals generated from digoxygenin-ddUTP-labeled probes were even for all samples and as strong as those obtained in MHC class II typing. These locus-specific primer sets derived from intron sequences provide an effective means to amplify genomic DNA which will facilitate PCR-based HLA class I typing methods. This will also allow HLA class I typing to be conducted with greater precision, at lower cost, and faster than previously described class I typing methodologies.  相似文献   

16.
The aim of our study was to define target determinants other than those coded for by the classical HLA-A, -B, -C or -D loci which were responsible for killing in CML. In one of the families studied, strong evidence was found for the existence of a determinant coded for within the HLA region. CML was restricted to targets carrying the classical HLA-Bw35 and Cw4 determinants but the targets were neither HLA-Bw35 nor Cw4 themselves. We therefore concluded that this new HLA determinant was either the product of a new locus closely associated with HLA-B or that it was a product of the classical HLA-B locus which has not been recognized by serology.  相似文献   

17.
HLA class I phenotyping was performed using T-lymphocyte populations isolated by immunomagnetic beads (IMBs) coated with monoclonal antibodies with specificity for CD2, CD4 or CD8. The results were compared to those obtained using density gradient-separated lymphocytes (PBL). The typing trays were read by the automated simultaneous double-fluorescence (SDF) technique previously established in our laboratory using an Astroscan 2100 system. The aims of the present study were to establish whether the advantages of IMB lymphocyte separation and automated plate reading by SDF were complementary and whether the results obtained by IMB-SDF and PBL-SDF were concordant. Similarity coefficients for paired results obtained by IMB-SDF and PBL-SDF varied between 0.825 using anti-CD8-coated IMBs and 0.914 using anti-CD4-coated IMBs with a consistent excess of stronger results observed with the PBL-SDF technique. The variations observed did not result in incorrect phenotype assignment but would significantly influence a cross-matching test. These results illustrate the feasibility of using IMB-separated lymphocytes for HLA phenotyping by SDF.  相似文献   

18.
We report HLA-A, -C, -B, -DRB1, -DQB1 and -DPB1 allele frequencies and estimated haplotype frequencies from 4514 healthy Norwegians who volunteered to participate in the Norwegian Bone Marrow Donor Registry. HLA genotyping was conducted on a Next Generation Sequencing platform. Data were analyzed using Arlequin and Pypop software. No significant deviations from Hardy-Weinberg Equilibrium were noted at any of the loci studied. We discuss the representability for the Norwegian population and argue that the presented HLA data could serve as a Norwegian reference panel.  相似文献   

19.
Abstract: We describe the application of RSCA for the high resolution typing of alleles encoded within the highly polymorphic HLA class I system. RSCA differs from other sequence based typing methodologies in that the HLA type is assigned on the basis of differences in DNA conformation between different HLA alleles. HLA class I locus specific PCR was performed on the sample to be HLA typed and the PCR product mixed with a fluorescent labelled locus specific reference strand. DNA molecules were allowed to reanneal and the duplexes formed were separated by non-denaturing PAGE. Only duplexes which possess the fluorescent labelled reference strand were detected using an automated DNA sequencer. The mobilities of duplexes formed for 131 HLA class I alleles have been calculated and HLA type assigned. The use of a laser scanning system for accurate detection of migrating DNA, in combination with specific software for data analysis and correction of gel variablility, make RSCA sufficiently robust to detect alleles differing by as little as one nucleotide, resulting in a simple and automated technique for high resolution HLA typing.  相似文献   

20.
设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号