HLA CLASS-II TYPING - DIRECT SEQUENCING OF DRB, DQB, AND DQA GENES
HLA CLASS-II TYPING - DIRECT SEQUENCING OF DRB, DQB, AND DQA GENES
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DOI：
10.1016/0198-8859(92)90056-s
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发表时间：
1992-02-01
期刊：
影响因子：
2.7
通讯作者：
RICH, SS
中科院分区：
文献类型：
--
作者：
SANTAMARIA, P;BOYCEJACINO, MT;RICH, SS
常规临床HLA II类分型主要基于血清学和细胞学方法。这些方法有许多缺点，导致分子分型方法的评估，包括限制性片段长度多态性研究和寡聚分型。我们在这里提出了一种替代的分子方法，基于序列的分型（SBT），允许直接确定所有HLA II类多态性基因的序列，从而提供了目前在这方面可能的最详细的信息。这里使用SBT提供的数据是基于使用有限数量的寡核苷酸对聚合酶链反应（PCR）扩增的DRB、DQB和DQA cDNA进行直接测序。设计寡核苷酸以允许同时测定任何杂合子中的等位基因序列以及表征DRB同种型复杂性。两种类型的扩增寡核苷酸（非保守和/或保守）用于DRB分型，这涉及最多四个同时进行的cDNA/PCR/测序反应。这些反应中的第一个仅使用保守的寡核苷酸，旨在检测任何给定杂合子中存在的所有不同的DRB转录本;其他三个反应使用非保守的寡核苷酸，旨在确保最复杂的DRB杂合子组合的明确解释。DQA 1和DQB 1序列的表征可以通过使用保守的寡核苷酸进行，并且每个locus.We只涉及一个反应应用SBT到43个纯合子细胞系和38个不同的杂合子组合，以前已血清学分型。在所有情况下，我们能够确定这些细胞系和受试者的DRB 1、DRB 3/4/5和/或DQB 1和DQA 1基因座的等位基因组成;我们的结果通过盲法方案分析，与血清学表型一致。SBT可以扩展到I类和III类基因，并且是自动化的。我们认为，这种策略值得进一步评估作为一种可能的HLA分型方法。
Routine clinical HLA class II typing is based largely on serological and cellular methods. These methods have many drawbacks that have led to the evaluation of molecular approaches to typing, including restriction fragment length polymorphism studies and oligotyping. We present here an alternative molecular approach, sequence-based typing (SBT), that allows direct determination of the sequences of all HLA class II polymorphic genes, thus providing the most detailed information currently possible in this regard. The data presented here using SBT are based on direct sequencing of polymerase chain reaction (PCR)-amplified DRB, DQB, and DQA cDNAs using a limited number of oligonucleotides. The oligonucleotides are designed to allow simultaneous determination of allelic sequences in any heterozygote as well as characterization of DRB isotypic complexity. Two types of amplification oligonucleotides (nonconserved and/or conserved) are used for DRB typing, which involves a maximum of four simultaneous cDNA/PCR/sequencing reactions. The first of these reactions only uses conserved oligonucleotides and is designed to detect all the different DRB transcripts present in any given heterozygote; the other three reactions use nonconserved oligonucleotides and are designed to ensure the unambiguous interpretation of the most complex DRB heterozygote combinations. Characterization of DQA1 and DQB1 sequences can be performed by using conserved oligonucleotides and only involves one reaction per locus.We have applied SBT to 43 homozygous cell lines and to 38 different heterozygote combinations that had previously been serologically typed. In all cases we were able to determine the allelic composition at DRB1, DRB3/4/5 and/or DQB1, and DQA1 loci of these cell lines and subjects; our results, analyzed by blind protocol, were consistent with the serological phenotypes. SBT can be extended to class I and class III genes and is automatable. We believe that this strategy deserves further evaluation as a possible HLA typing method.