HLA-B typing by allele separation followed by direct sequencing.
HLA-B typing by allele separation followed by direct sequencing.
复制标题
通过等位基因分离进行 HLA-B 分型，然后直接测序。
DOI：
10.1111/j.1399-0039.1997.tb02764.x
复制
发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Watkins,DI
中科院分区：
文献类型：
--
作者：
Eberle,M;Knapp,LA;Iwanaga,KK;Domanico,MJ;Aiyer,K;Watkins,DI
由于人类白细胞抗原基因座具有巨大的等位基因多样性，因此很难设计一种明确的等位基因分型方法。在这里，我们描述了一种用于人类白细胞抗原等位基因直接测序的技术。首先，对RNA进行位点特异性逆转录后，用聚合酶链式反应扩增出人类白细胞抗原等位基因。然后使用变性梯度凝胶电泳法(DGGE)分离等位基因，DGGE根据DNA片段的序列组成分离DNA片段。从凝胶中取出扩增产物，洗脱的DNA被重新扩增并直接测序。将得到的序列与已公布的人类白细胞抗原-B序列数据库进行比对，并进行了初步的等位基因分配。从理论上讲，这种方法足以对118个已知的人类白细胞抗原等位基因中的92个进行分型。其余26个等位基因中的大多数在外显子2的开始处存在差异，外显子2是DGGE分离的PCR产物之外的区域。因此，我们使用该区域的杂合测序来鉴定这26个等位基因中的19个，将分辨率提高到111个等位基因。使用这项技术，我们分析了来自几个不同来源的永生化细胞系和血液样本。从第十届国际组织相容性研讨会(IHWS)获得了9个永生化细胞系，其中9个来自土著人民。此外，从一个献血者小组中获得了25个血液样本，这些样本以前被证明是难以使用血清学技术进行分型的。总之，使用DGGE分离等位基因然后直接测序的新方法，我们从57个个体中分型了52个不同的等位基因，涵盖了40个血清学特异性。
Due to the enormous allelic diversity of theHLA‐Blocus, it has been difficult to design an unambiguous molecular typing method for the alleles at this locus. Here we describe a technique for the direct sequencing ofHLA‐Balleles. Initially,HLA‐Balleles were PCR‐amplified after locus‐specific reverse transcription of RNA. Alleles were then separated using denaturing gradient get electrophoresis (DGGE), which separates DNA fragments based on their sequence composition. Amplification products were excised from the gel and eluted DNA was reamplifled and directly sequenced. The derived sequences were aligned to a database of publishedHLA‐Bsequences, and an initial allele assignment was made. This approach was theoretically sufficient to type 92 of the 118 knownHLA‐Balleles. The majority of the remaining 26 alleles contain differences at the beginning of exon 2, a region outside the DGGE‐separated PCR products. Therefore, we used heterozygous sequencing of this region to identify 19 of these 26 alleles, raising the resolution power to 111 alleles. Using this technique, we analyzed immortalized cell lines and blood samples from several different sources. Nine immortalized cell lines were obtained from the 10th International Histocompatibility Workshop (IHWS) and nine were derived from aboriginal peoples. Additionally, 25 blood samples were acquired from a panel of donors previously shown to be difficult to type using serological techniques. Altogether, using this new method of allele separation by DGGE followed by direct sequencing, we typed 52 different alleles from 57 individuals, covering 40 serological specificities.