ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00001.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00002.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYDNA‐restriction fragment length polymorphism (RFLP) typing is a well established and standard technique for identification of HLA‐DR and DQ allotypes. We present a detailed review of methods for RFLP analysis and a scheme for interpretation of results. This scheme permits the routine identification of HLA‐DR and DO allotypes using the restriction endonucleasesTaqland eitherHindIIIo

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00003.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThe polymerase chain reaction (PCR) has made the technique of sequence‐specific oligoncucleotide (SSO) typing fast, accurate and very sensitive. These combined techniques are an ideal tool for analysing the complex patterns of polymorphism seen throughout the HLA complex. The success of the technique relies heavily on accurate and specific amplification of the DNA under study. This paper considers the principles behind the PCR amplification technique and discusses the factors which lead to optimal amplification. Primer design is discussed and a variety of sources of target DNA considered. Precautions designed to prevent contamination are discussed. Reaction components are considered both in isolation and as part of the complete reaction. Finally, a complete PCR protocol is suggested. The paper is illustrated with examples of HLA class II amplificatio

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00004.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYA simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence‐based HLA typing strategies, like oligonucleotide probe hybridization, is that they revealhowandwheretwo alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non‐Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all ‘new’ alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available wouldnotbe revealed by oligonucleotide typing.With the PCR primers and probes described here, 7 DQA1 alleles,* 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)‐labelled oligonucleotide probes are stable (>2 years when stored at 4°C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich,et al., manuscript in preparation) and>1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin‐HRP conjugates and either chromogenic or chemiluminescent substrates. This method offers the simplest and most rapid approach to the HLA typing of clin

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00005.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYSeveral sequence‐specific oligonucleotides (SSO) were designed based upon available sequences of HLA‐DRB 1 genes which are associated with HLA‐DR serotypes. Targets for these SSO were polymerase chain reaction (PCR) products using primers specific for the second exon of DRB genes. In view of practicality, a hybridization system was developed which was independent of temperature or composition of the hybridization buffers, hence uniformly applicable to all SSO, yet allowing discrimination based upon one nucleotide mismatch only. The SSO typing results were compared with HLA serological typing and a high correlation was observed

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00006.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYPolymerase chain reaction (PCR) amplification of the second exon of DRB genes from genomic DNA is described. Sequence‐specific oligonucleotide (SSO) probes have been designed to allow for the typing of DRB alleles. The methods are described in detail and the results of a trial on both homozygous typing cells and restriction fragment length polymorphism (RFLP)‐typed local caucasoid controls are given. The ease of use of this form of DRB typing is emphasized and potential complications are discus

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00007.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00008.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYThirty‐seven DR4‐positive patient‐unrelated bone marrow donor pairs previously DR/DQ restriction fragment length polymorphism (RFLP) typed and tested in mixed lymphocyte culture (MLC), have been DR4‐Dw subtyped retrospectively using sequence specific oligonucleotide probes. We found that DR4‐Dw subtyping substantially increased the accuracy of pre‐MLC matching and could potentially accelerate donor searches by avoiding unnecessary MLC tests on Dw‐mism

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00009.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY

摘要:

SUMMARYSerological identification of the HLA‐DQw1(w5)‐associated or HLA‐DQw3(w7)‐associated DR ‘Br’(DRB1*0103) allele cannot be accomplished in the presence of a second DQw1(w5)‐positive or DQw3(w7)‐positive haplotype, respectively. DNA‐restriction fragment length polymorphism (RFLP) analysis assists in identification of DR ‘Br’, though not in the presence of DR1. We describe an alternative or complementary method for identification of DR ‘Br’ using two oligonucleotide probes which target

ISSN:1744-3121    

DOI:10.1111/j.1744-313X.1991.tb00010.x

出版商:Blackwell Publishing Ltd    

年代:1991

数据来源: WILEY