Sequencing of HLA class II genes based on the conserved diversity of the noncoding regions: sequencing based typing of HLA-DRB genes

被引:186
作者
Kotsch, K [1 ]
Wehling, J [1 ]
Blasczyk, R [1 ]
机构
[1] Hannover Med Sch, Dept Transfus Med, D-30625 Hannover, Germany
来源
TISSUE ANTIGENS | 1999年 / 53卷 / 05期
关键词
HLA-DRB; sequencing based typing; lineage-specific intron diversity;
D O I
10.1034/j.1399-0039.1999.530505.x
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
In this paper, we present a novel sequencing based typing strategy for the HLA-DRB1, 3, 4 and 5 loci. The new approach is based on a group-specific amplification from intron 1 to intron 2 according to the serologically-defined antigens. For this purpose, we have determined the 3' 500 bp-fragment of intron 1 and the 5' 340 bp-fragment of intron 2 of all serological antigens and their most frequent subtypes. We discovered a remarkably conserved diversity characterized by lineage-specific sequence motifs This lineage-specificity of non-coding motifs in the 1st and 2nd intron offered the possibility to establish a clear serology-related amplification strategy, The method allows the complete analysis of the 2nd exon and the definition of the cis/trans linkage of sequence motifs by intron-mediated polymerase chain reaction (PCR)-based separation of the haplotypes in nearly all serologically heterozygous samples. In particular, the non-coding variabilities between the DR52-associated DRB1 groups made the independent amplification possible. Thus, compared to the standard procedures using exon-based amplification primers, the groups DR3, DR12, some DR13 alleles (1301, 1302) and the DR14 group could be amplified by specific primer mixes. The DR8 could be amplified with an individual primes mix not co-amplifying the DR12. The DR11 and DR13 did not show any individual motif in intron 1 or intron 2. In order to achieve a separate amplification, they had to be amplified by multispecific primer mixes (DR3/11/13/14; DR3/11/13 or DR11/13/14) excluding the other haplotype. Thus, exclusively the alleles in rare DR11,13 heterozygosities without a DRB1*1301 or 1302 could not be amplified separately. Fourteen primer mixes are used to amplify the DR1-14, and 6 primer mixes for the specificities DR51-53. The sequence homology of the 3' end of intron 1 facilitated the application of only three different sequencing primers for all DRB alleles.
引用
收藏
页码:486 / 497
页数:12
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