A simple and rapid method for HLA-DQA1 genotyping by digestion of PCR-amplified DNA with allele specific restriction endonucleases

The second exon of the HLA-DQA1 genes was selectively amplified from genomic DNAs of 72 HLA-homozygous B cell lines by the polymerase chain reaction (PCR). Amplified DNAs were digested with HaeIII, Ddel, ScrFI, FokI and RsaI, which recognize allelic sequence variations in the polymorphic segments of the DQA1 second exon, and then subjected to electrophoresis in polyacrylamide gels. Eight different polymorphic patterns of restriction fragments were obtained, and seven were identical to patterns predicted from the known DNA sequences, correlating with each HLA-DQw type defined by serological typing. The remaining one pattern cannot be explained from the sequence data, suggesting the presence of a novel DQA1 allele at the nucleotide level. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DQ types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes and can be extended and applied to HLA-DR, -Dw DP typing.     

A simple and rapid method for HLA-DRB and -DQB typing by digestion of PCR-amplified DNA with allele specific restriction endonucleases

The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, which we previously reported as an efficient and convenient typing technique for accurate definition of the HLA-DQA1 and -DPB1 alleles, is now extended and applied to HLA-DRB and -DQB typing. The second exon of the HLA-DRB (B1 and B3 or B4) and DQB (B1 and B2) genes was selectively amplified from genomic DNAs of 70 HLA-homozygous B cell lines by PCR. Amplified DNAs were digested with the restriction endonucleases, which can recognize allelic variations specific for HLA-DR, -DQ, and -Dw allospecificities and then subjected to electrophoresis in polyacrylamide gel. Of DRB genes, FokI, HinfI, HhaI, HphI, KpnI and SacII were selected and the 20 different polymorphic patterns of the restriction fragments thus obtained were found to correlate with each HLA-DR and -Dw type defined by serological and cellular typing. Of the DQB genes, FokI, HaeIII, HhaI, RsaI and Sau3AI produced nine different polymorphic patterns of the restriction fragments, correlating with the HLA-DQ and -Dw types. This PCR-RFLP method provides a simple and rapid technique for accurate definition of the HLA-DR, -DQ and -Dw types at the nucleotide level, eliminating the need for radioisotope as well as allele specific oligonucleotide probes.     

HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides

HLA-DP genotyping was performed using dot-blot analysis with synthetic oligonucleotide probes. Fourteen probes were designed based on the known sequence variations in the polymorphic segments of the DP beta second exon. Each probe was tested against genomic DNA amplified by the polymerase chain reaction, using DP beta-specific primers. A total of 45 HLA homozygous B-cell lines, selected from the Tenth International Histocompatability Workshop and pretyped for the known DP omega specificities, were analyzed. Different hybridization patterns were found for each DP omega specificity. The oligonucleotide hybridization performed on DP omega-negative B-cell lines gave a pattern distinct from those of known DP omega specificities, indicating the presence of novel DP allelic sequences. The use of sequence-specific oligonucleotides combined with DNA amplification allows a simple and reliable genotyping of DP antigens.     

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers

Abstract: We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB 1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplifed by DQwl group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQwl-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB 1*0602 and DQB 1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB 1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.     

HLA-DQB1 genotyping by a modified PCR-RFLP method combined with group-specific primers.

We previously reported a simple technique for HLA-DQB genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). However, this method has some problems in that some heterozygotes cannot be discriminated from each other. Furthermore, concomitantly amplified product derived from the DQB2 gene by the primers used previously also obstructs precise DQB1 genotyping. To resolve these problems, we have developed two different pairs of specific primers for selective amplification of the DQB1 gene and also used restriction endonucleases which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA-DQB1 alleles, making reading of RFLP band patterns much easier. The second exon of the DQB1 gene was selectively amplified by DQw1 group-specific primers and/or DQw2,3,4 group-specific primers using genomic DNAs from 70 HLA-homozygous B-cell lines and 50 healthy Japanese. Of the seven DQw1-associated DQB1 alleles, six alleles could be defined by digestion of 6 restriction enzymes, although DQB1*0602 and DQB1*0603 could not be discriminated from each other because of unavailability of suitable enzymes. Similarly, all of the six DQw2,3,4-associated DQB1 alleles could be defined by digestion of 5 restriction enzymes. Using this modified PCR-RFLP method, complete DQB1 genotyping of all heterozygotes is possible except for discrimination between DQB1*0602 and 0603. Thus this method is simpler and more practical for a routine DNA typing than the PCR-SSO method or our previous PCR-RFLP method.     

Severe acute graft-versus-host disease by HLA-DPB1 disparity in recombinant family of bone marrow transplantation between serologically HLA-identical siblings: An application of the polymerase chain reaction—restriction fragment length polymorphism method

It has remained to be established that matching of the HLA-DP antigen plays a key role in bone marrow transplantation (BMT), mainly due to the difficulty of the primed lymphocyte test (PLT) method for DP typing. We previously reported an efficient technique for HLA class II genotyping by digestion of polymerase chain reaction (PCR)-amplified genes with restriction fragment length polymorphisms (RFLP) endonucleases (PCR-RFLP method). DNAs from 46 recipients and corresponding donors in serologically HLA-identical sibling-BMT cases were DP typed by this PCR-RFLP method. Of the 46 cases, five (10.9%) were genetically DP mismatched (recombinant frequency between the DR-DQ and DP subregions was at least 2.7% per meiosis), providing an important opportunity to look at the effect of the disparity only seen in the DP antigen on BMT. Three of the four DP-mismatched BMT cases that could be evaluated developed severe acute graft-versus-host disease, suggesting that DP disparity played an important role in BMT.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping

Abstract: We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacryl-amide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, Fokl, MboII and Mnll can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, Fokl, Ddel, BsaJI, BssHII, Cfr13I, Rsal, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

Modified PCR-RFLP method for HLA-DPB1 and -DQA1 genotyping.

We previously developed a new technique for HLA class II genotyping by digestion of polymerase chain reaction-amplified genes with restriction endonucleases (PCR-RFLP method). This PCR-RFLP method is an efficient and convenient typing technique for class II alleles. However, small fragments or bands located close to each other on polyacrylamide gels sometimes prevent precise analysis of the RFLP bands. Furthermore, the restriction enzymes we have reported in the previous papers are not sufficient to identify the genotypes of all heterozygous individuals. Here, we report an improved PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or, alternatively, no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. Each second exon of the HLA-DQA1 or -DPB1 gene was selectively amplified from genomic DNAs of 70 HLA-homozygous B-cell lines and 100 healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA. ApaLI, HphI, BsaJI, FokI, MboII and Mn1I can discriminate eight alleles of the DQA1 gene. Similarly 19 alleles of the DPB1 gene can be discriminated with Bsp1286I, FokI, DdeI, BsaJI, BssHII, Cfr13I, RsaI, EcoNI, and AvaII enzymes. This modified PCR-RFLP method can be successfully applied to heterozygotes. Thus, the method is technically simpler and more practical for routine HLA typing work than our previous PCR-RFLP method.     

HLA-DRB1 genotyping by modified PCR-RFLP method combined with group-specific primers

We previously introduced HLA-DQA1, -DPB1 and DQB1 genotyping with the modified PCR-RFLP method using some informative restriction enzymes which have either a single cleavage site or alternatively no cleavage site in the amplified DNA region, depending on the HLA alleles, making reading of RFLP band patterns much easier. In this study, 43 HLA-DRB1 alleles, excluding DRB1*1103 and *1104 for which no restriction enzymes are available to distinguish each from the other, could be defined by this modified PCR-RFLP method combined with 7 pairs of group-specific primers. It is impossible to distinguish DRB1*0701 and DRB1*0702 as they are identical for the second exon of DRB1. For DR1-DRB1, DR2-DRB1, DR4-DRB1, DR7-DR1, DR9-DRB1, DRw10-DRB1 or DRw52 associated antigens (DR3, w11, w12, w13, w14, and DRw8)-DRB1 gene amplification, the second exon of the DRB1 gene was selectively amplified using each group-specific primer from genomic DNAs of 70 HLA-homozygous B-cell lines and healthy Japanese by PCR. Amplified DNAs were digested with restriction endonucleases and then subjected to electrophoresis assaying simply for cutting, or no cutting, of the DNA, although some alleles can be distinguished only after examination of RFLP band patterns generated and in some cases using double digestion technique with two restriction enzymes. This modified PCR-RFLP method can be successfully applied to all possible DRB1 heterozygotes, despite the fact that 15 pairs of heterozygotes among them cannot be distinguished theoretically by the PCR-SSO method, because the PCR-RFLP method can tell whether two polymorphic sites are linked to each other (cis position) or located on a different chromosome (trans position) by checking the length of RFLP bands generated with double digestion. Thus, the PCR-RFLP method is technically simple, practical and inexpensive for determination of the HLA-DRB1 alleles for routine HLA typing work.     

DNA typing of the HLA-A gene: population study and identification of four new alleles in Japanese

With the use of polymerase chain reaction (PCR) and sequence-specific oligonucleotide probe (SSOP), we established a DNA typing method of the HLA - A locus. A pair of primers to amplify the highly polymorphic region of HLA-A gene including exon 2 and exon 3 was designed and the amplified DNAs were hybridized with 91 types of ³²P labeled SSOPs. This method allowed discrimination of all known HLA-A alleles except for two combinations, A*0201 or A*0209 and A*0207 or A*0215N, which have identical sequences in exon 2 and exon 3. Another pair of primers was designed for amplification of exon 4 and the PCR products were hybridized with 5 SSOPs to distinguish A*0201 and A*0207 from A*0209 and A*0215N, respectively. In this study, 81 B-lymphoblastoid cell lines (BLCL) homozygous for HLA and 553 unrelated healthy Japanese individuals were determined for their HLA-A genotypes. Based on the genotyping results, frequency of HLA-A alleles and linkage disequilibrium between HLA-A and HLA-B in the Japanese population were investigated. In addition, four new HLA-A alleles were identified and their nucleotide sequences in exon 2 and exon 3 were determined to confirm the typing results.     

Complete HLA-A DNA typing using the PCR-RFLP method combined with allele group- and sequence-specific amplification

We have established a practical method of complete high-resolution typing for all HLA-A alleles using the polymerase chain reaction (PCR)-restriction fragment-length polymorphism (RFLP) technique combined with allele group- and sequence-specific amplification. The second and third exons of the HLA-A gene, in which most allelic variations are observed, were separately amplified by PCRs with 3 and 4 group-specific primer pairs, respectively. Each PCR-amplified product was digested by allele-specific restriction endonucleases and then subjected to electrophoresis on a 10% polyacrylamide gel. In this way, 62 out of 79 HLA-A alleles could be discriminated by the RFLP patterns derived from the genetic polymorphism in the exon 2 and 3 domains. The remaining 17 alleles could be defined unequivocally by either PCR-RFLP analysis after exon 4 amplification or PCR analysis with sequence-specific primers (SSP). By this method, complete HLA-A genotyping for all homozygous and heterozygous combinations can be accomplished, establishing technically simple, economical and practical routine typing of the HLA-A gene, especially for small samples.     

Development of an ELA-DRA gene typing method based on pyrosequencing technology

The polymorphism of equine lymphocyte antigen (ELA) class II DRA gene had been detected by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and reference strand-mediated conformation analysis. These methodologies allowed to identify 11 ELA-DRA exon 2 sequences, three of which are widely distributed among domestic horse breeds. Herein, we describe the development of a pyrosequencing-based method applicable to ELA-DRA typing, by screening samples from eight different horse breeds previously typed by PCR-SSCP. This sequence-based method would be useful in high-throughput genotyping of major histocompatibility complex genes in horses and other animal species, making this system interesting as a rapid screening method for animal genotyping of immune-related genes.     

HLA class II specificities and haplotypes in South Africa detected using polymerase chain reaction and sequence-specific oligonucleotide typing.

DNA sequencing of HLA class II alleles has revealed a degree of polymorphism much greater than was expected on the basis of the standard serological typing methods. Amplification of the polymorphic second exon of the class II genes using the polymerase chain reaction, followed by hybridization with sequence-specific oligonucleotide probes, allows the unambiguous identification of alleles which could not be detected previously. Using the protocols of the Eleventh International Histocompatibility Workshop, we have applied this procedure for the typing of several individuals and their families with suspected alleles that had been observed using serology, cellular typing and restriction fragment length polymorphism (RFLPs). These included an allele related to DRw8 and DRw14, which has only been observed in the mixed ancestral South African population. In addition, unusual combinations of class II genes forming unique haplotypic associations were seen.     

HLA class II specificities and haplotypes in South Africa detected using polymerase chain reaction and sequence-specific oligonucleotide typing

Abstract: DNA sequencing of HLA class II alleles has revealed a degree of polymorphism much greater than was expected on the basis of the standard serological typing methods. Amplification of the polymorphic second exon of the class II genes using the polymerase chain reaction, followed by hybridization with sequence-specific oligonucleotide probes, allows the unambiguous identification of alleles which could not be detected previously. Using the protocols of the Eleventh International Histocompatibility Workshop, we have applied this procedure for the typing of several individuals and their families with suspected alleles that had been observed using serology, cellular typing and restriction fragment length polymorphism (RFLPs). These included an allele related to DRw8 and DRwl4, which has only been observed in the mixed ancestral South African population. In addition, unusual combinations of class II genes forming unique haplotypic associations were seen.     

Polymerase chain reaction--single-strand conformation polymorphism analysis of polymorphism in DPA1 and DPB1 genes: a simple, economical, and rapid method for histocompatibility testing.

A new technical trial was carried out to detect polymorphism in HLA-DP genes, based on the diversity in electrophoretic mobility of single-stranded DNA (single-strand conformation polymorphism, SSCP). Genomic DNAs from 31 cell lines homozygous for 2 and 14 different DPA1 and DPB1 alleles, respectively, and from peripheral blood cells of a normal individual homozygous for another DPB1 allele were subjected to polymerase chain reaction (PCR) to amplify the polymorphic exon 2 of DPA1 or DPB1 genes. The PCR samples were denatured by heating in the presence of formamide to obtain single-stranded DNA, electrophoresed in a neutral polyacrylamide gel, and visualized by silver staining. Allelic differences were detected by the distinctive electrophoretic pattern of each single strand, depending on the sequence-specific conformation. Fifteen DPB1 alleles showed 11 distinct electrophoretic patterns, leaving four allelic combinations not distinguished. These four allelic combinations could be further distinguished by using another couple of primers in PCR, with which a part of the exon was amplified, and by subsequent SSCP analysis. The use of four pairs of primers in PCR allowed for discrimination of all the 15 DPB1 alleles tested. Two allelic differences in exon 2 of DPA1 gene could be clearly demonstrated. In addition, putative new alleles of DPA1 and DPB1 genes were detected by SSCP analyses. The PCR-SSCP analysis is simple and rapid, requires neither radioactive materials nor restriction enzymes, and is expected to be a useful tool for investigating the fine HLA-matching required for clinical transplantation of organs.     

HLA-DPB1 typing by polymerase chain reaction amplification with sequence-specific primers

DPB1 is the second most polymorphic class II locus with currently 84 recognized alleles, i.e. DPB1*0101 to DPB1*8101. Most of the alleles have been described during the last few years using oligonucleotide and sequencing techniques and relatively little is known about the role and importance of the polymorphic residues as regards to the function of DP molecules. In the present study, polymerase chain reaction (PCR) primers were designed for identification of all the phenotypically different DPB1 alleles by PCR amplification with sequence-specific primers. Forty-eight standard genomic PCR reactions per sample were performed in order to achieve this resolution. Unique amplification patterns were obtained in 2983 of 3160 (94.4%) possible genotypes. The primers were combined so that only very rare genotypes gave rise to ambiguous patterns. Sixty-four Histocompatibility Workshop cell lines and 150 DNAs provided by the UCLA DNA exchange were investigated by the DPB1 primer set. All typing results were conclusive. Analysis of the distribution of DPB1 alleles was performed in 200 Caucasian samples, 100 African samples and 40 Oriental samples. The population study by the DPB1 PCR-SSP method showed a characteristic distribution of HLA-DPB1 alleles. Each ethnic group had one, or two, frequent DPB1 allele(s) and the frequency of homozygotes was high, suggesting that balancing selection does not appear to be affecting the evolution of the DPB1 locus.     

How many probes are needed for HLA-DPB1 typing with sequence-specific oligonucleotide probes? A theoretical approach using computer simulation

HLA-DP genotyping with sequence-specific oligonucleotides is used to detect known sequence variations in the polymorphic segments of the DPB1 second exon. This approach is a valuable method replacing the tedious cellular definition of DP polymorphism. We have addressed, by computer simulation, the question: what is the minimum number of probes needed to provide an unambiguous assignment of HLA-DP alleles by genotyping of heterozygous individuals? We were able to reduce the number of probes in a set defining the presently known 22 different alleles and most of the heterozygous combinations to 18 probes. Only two pairs of allelic combinations cannot be distinguished by this method, neither with our optimized set of probes nor with any larger set comprising probes of reasonable length. This is because two pairs of alleles may be the result of a reciprocal genetic exchange. These two pairs, however, could be distinguished by family analysis, direct sequencing, or DNA amplification using specific primers chosen from the polymorphic ends of the DPB1 second exon.     

使用PCR—SSP方法检测HLA—DR抗原

目的　采用顺序特异引物聚合酶链反应 (PCR -SSP)建立人类白细胞抗原DR位点的DNA分型方法 .方法　合成 2 9个特异性引物和 1对阳性对照引物 ,组成 2 0个PCR反应用于DR位点 ,建立一步法PCR -SSP .结果　所有样本PCR -SSP基因分型获得成功 ,分型结果经标准DNA ,限制性核酸内切酶分析证实符合 ,特异性和重复性 10 0 % .结论　PCR -SSP检测HLA -DR的方法具有快速、准确、特异性高等优点 ,适合临床应用 .     

The human complement C8G gene, a member of the lipocalin gene family: polymorphisms and mapping to chromosome 9q34.3

Complement component C8 is a plasma glycoprotein consisting of three nonidentical polypeptide chains (α, β, γ) which are encoded by three separate genes (C8A, C8B, C8G). The γ chain whose functional role remains undefined is not related to any other complement protein but is a member of the lipocalins, a family of proteins that bind small hydrophobic ligands. The present report describes the first known polymorphisms for the human C8G gene, namely one polymorphic site in exon 1 (207T/G) and two polymorphic sites in intron 1 (213 + 37G → A; 213 + 65del3). Specific typing can be performed using simple polymerase chain reaction-based assays. C8G genotyping in eight CEPH reference families demonstrated that C8G is closely linked to a series of marker loci located in the most telomeric region of chromosome 9q. Multipoint analysis placed C8G with 1000:1 support distal to D9S207. C8G is thus located at 9q34.3. Remarkably, this chromosomal region contains at least four other lipocalin genes.     

Establishment of a sequence-based typing system for BoLA-DRB3 exon 2

A rapid, high-resolution sequence-based typing (SBT) system for BoLA-DRB3 exon 2 was developed. Amplification of the entire exon was achieved by a fully nested PCR with locus-specific primers and sequencing was performed directly on the PCR product. Heterozygous sequence data were obtained by automated sequence analysis of both alleles. Forward and reverse sequence data were assembled to improve identification of all heterozygous positions. Specific software (Haplofinder, Roslin Institute Software, Roslin, UK) was designed for allele assignment. Fifty-four females from a Holstein-Charolais resource herd cross, their 12 sires and five unrelated Holstein animals were used to establish the method. In parallel, these animals were typed by DRB3 polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to confirm the results. Polymerase chain reaction-RFLP analysis defined 15 known types in the 71 animals, while SBT of the same animals showed 19 known alleles. Subsequently, 72 more animals from the same resource herd were typed by the established SBT method without PCR-RFLP typing. This SBT strategy and the Haplofinder software can be applied to the analysis of any polymorphic locus for which suitable locus-specific primers and allelic sequences are available.