Hla‐dr, Do and Dp Typing Using PCR Amplification and Immobilized Probes

Abstract

A 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 reveal how and where two 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 would not be 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 (greater than 2 years when stored at 4 degrees 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 Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure.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.(ABSTRACT TRUNCATED AT 400 WORDS)

Screening the genome for rheumatoid arthritis susceptibility genes: A replication study and combined analysis of 512 multicase families

“…Broad-level HLA-DRB1 typing and high-resolution DRB1‫40ء‬ typing were accomplished by initial polymerase chain reaction (PCR) amplification of groups of alleles (all DRB1 alleles for broad-level typing, and group-specific amplification for DRB1‫40ء‬ alleles) using biotinylated PCR primers, followed by hybridization to immobilized sequence-specific oligonucleotide probes in a linear array format. Positive hybridization reactions were detected using a streptavidinhorseradish peroxidase conjugate and a soluble colorless substrate, 3,3Ј,5,5Ј-tetramethylbenzidine (15). A computer algorithm based on the sequence-specific oligonucleotide probe hybridization pattern and the Anthony Nolan 1999 HLA sequence database (available at the World Wide Web site http://www.ebi.ac.uk/imgt/hla/nomen.html) were used to assign genotypes to each sample.…”

High-density SNP analysis of 642 Caucasian families with rheumatoid arthritis identifies two new linkage regions on 11p12 and 2q33

“…Positive hybridization reactions were detected using a streptavidin-horseradish peroxidase conjugate and a soluble colorless substrate TMB (3,3 0 ,5,5 0 -tetramethylbenzidine). 59 A computer algorithm based on the sequence specific oligonucleotide probe hybridization pattern and the Anthony Nolan 1999 HLA sequence database (http://www.ebi.ac.uk/imgt/hla/) was used to assign genotypes. The following alleles detected were classified as 'SE' positive: DRB1*0101, 0102, 0104, 0105, 0401, 0404, 0405, 0408, 0409, 1001, 1402 and 1406.…”

Revisao dos dados citogeneticos em especies da família Hylidae (amphibia, anura): resultados e perspectivas

“…Também se confi rmou a origem fetal das células mesenquimais coletadas do líquido amniótico e cultivadas in vitro por meio da tipagem molecular dos antígenos leucocitários humanos. (ERLICH, et al, 1991 A utilização das CTM no reparo tecidual é estudada há tempos (BRUDER, et al, 1993), porém, o potencial dessas células proporciona muitas outras aplicações experimentais. Seu potencial de diferenciação em diversos tecidos, suas propriedades imunossupressoras e a relativa facilidade de manuseio in vitro fazem das CTM uma ferramenta importante para, entre outras coisas, a engenharia tecidual, tratamento de doenças degenerativas e estudo de novos fármacos.…”