HLA DRB TYPING USING DENATURING GRADIENT GEL ELECTROPHORESIS & SEQUENCING

骨髓需要高分辨率的人类白细胞抗原-DRB配型 无血缘关系的供者和受者之间的移植以及 人类白细胞抗原DRB新等位基因的鉴定。高度可变的秒 所有HLA-DRB1、-DRB3、-DRB4、-DRB5、-DRB6和-DRB7等位基因的外显子均为 使用一对通用的DRB特异引物进行扩增 用DGGE分离等位基因。然后从插头中重新扩增DNA 从凝胶中移除，这些等位基因的序列 使用基于荧光的测序和等位基因分配确定 软件。结果证实了该分型方法的有效性 通过鉴定17名个体的人类白细胞抗原-DRB等位基因 以PCR-SSP和/或克隆和测序技术为特征。我们 在这17个无关的人中发现了34个不同的HLA-DRB等位基因 个人。我们的分析揭示了人类白细胞抗原-DRB1等位基因 用聚合酶链式反应-单链构象多态性分型技术鉴定。另外， 检测到了人类白细胞抗原-DRB3、-DRB4和-DRB5基因座的等位基因。 而传统的人类白细胞抗原-DRB配型方法提供的信息有限 或需要使用多个寡核苷酸引物或探针，我们的 技术提供了一种可靠、特定和相对快速的方法 鉴定所有用于高分辨率组织分型的人类白细胞抗原-DRB等位基因。 前言：目的：描述一种明确的鉴别方法。 用聚合酶链式反应(PCR)检测HL A-DRB等位基因，变性 梯度凝胶电泳法(DGGE)及其发展方向 现在可以用这项技术来确定患者的DRB1等位基因 正在接受骨髓移植。关键词交叉反应， DGGE、DNA、人类白细胞抗原-DRB、聚合酶链式反应、测序、分型

High-resolution HLA-DRB typing is required for bone marrow transplantation between unrelated donors and recipients and also for identification of novel HLA-DRB alleles. The highly variable second exon of all HLA-DRB1, -DRB3, -DRB4, -DRB5, -DRB6 and -DRB7 alleles was amplified using a single pair of generic DRB-specific primers and alleles were separated by DGGE. DNA was then reamplified from plugs removed from the gel and the sequences of these alleles were determined using fluorescent-based sequencing and allele-assignment software. RESULTS The validity of this typing procedure was confirmed by identification of HLA-DRB alleles for 17 individuals previously characterized by PCR-SSP and/or cloning and sequencing techniques. We identified 34 different HLA-DRB alleles in these 17 unrelated individuals. Our analysis revealed HLA-DRB1 alleles which had not been identified using the PCR-SSP typing technique. Additionally, alleles from the HLA-DRB3, -DRB4 and -DRB5 loci were identified. Whereas traditional HLA-DRB typing methods provide limited information or require the use of multiple oligonucleotide primers or probes, our technique provides a reliable, specific and relatively rapid way of identifying all HLA-DRB alleles for high-resolution tissue typing. OBJECTIVE: To describe a method for the unambiguous identification of HLA-DRB alleles using the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE) and direct FUTURE DIRECTIONS This technique can now be used to define the DRB1 alleles of patients undergoing bone marrow transplantation. KEY WORDS cross-reactivity, DGGE, DNA, HLA-DRB, PCR, sequencing, typing