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  • Title: New strategies for efficient typing of HLA class-II loci DQB1 and DRB1 by using Pyrosequencing.
    Author: Entz P, Toliat MR, Hampe J, Valentonyte R, Jenisch S, Nürnberg P, Nagy M.
    Journal: Tissue Antigens; 2005 Jan; 65(1):67-80. PubMed ID: 15663743.
    Abstract:
    The characterization of genetic risk factors for complex diseases located on chromosome-6 frequently requires human leucocyte antigen (HLA) genotyping of large patient cohorts. Currently available methods do not support high-throughput HLA typing beyond the major allele group level. We, thus, developed a high-throughput approach for the HLA-DQB1 and HLA-DRB1 loci that is based on Pyrosequencing. Pyrosequencing offers a higher degree of automation than direct sequencing or oligotyping. Using a dispensation order optimized for the particular HLA locus, rapid group typing and fine resolution can be achieved. We implemented the method for two important HLA loci--DQB1 and DRB1. The HLA-DQB1 typing method comprises the following steps: splitting the potential alleles after a generic polymerase chain reaction (PCR) amplification into groups with a first Pyrosequencing reaction and resolving the split allele groups by means of five further Pyrosequencing reactions. The HLA-DR gene family is known to be the most polymorphic one in the HLA class-II region because of a large number of DRB1 alleles. Because of this complex nature, HLA-DRB1 typing was performed by means of a combination of sequence-specific PCR typing and Pyrosequencing. HLA-DQB1 typing and HLA-DRB1 typing were performed successfully by using standard DNA samples with the help of known HLA genotypes and in a blind study by using the samples from the Deutscher Zell Austausch 2002 and 2003. The approach was optimized and was practically tested for genotyping in disease association studies. Our well-elaborated Pyrosequencing-based protocols offer a new alternative to the existing HLA class-II typing methods and represent a convenient and economic solution, a unique combination of high accuracy with high-sample throughput.
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