基因组DNA高通量提取技术及其在骨髓库捐献者样本HLA基因分型中的应用  

High Through-put Genomic DNA Isolation Technique and Its Application in HLA Genotyping for Samples from Bone Marrow Donor Program

作　　者：王大明[1] 唐斯[1] 李桢[1] 程曦[1] 高素青[1] 邓志辉[1] 

机构地区：[1]深圳市血液中心,广东深圳518035

出　　处：《中国实验血液学杂志》2009年第5期1265-1268,共4页Journal of Experimental Hematology

摘　　要：本研究旨在建立从全血EDTA抗凝样本中高通量提取基因组DNA的有效方法,以常规应用于Luminexr SSO HLA流式磁珠基因分型。使用2ml深孔板和TECAN DNA全自动工作站,从288份骨髓捐献者样本中提取基因组DNA;提取的DNA样本用紫外分光光度仪测定其浓度和纯度,并采用琼脂糖凝胶电泳检测DNA的完整性;DNA样本用Onelambdar SSOHLA-A、B和DRB1基因分型试剂盒进行PCR扩增、分子杂交和Luminex流式磁珠分析仪检测,统计分析每一DNA样本HLA-A、B和DRB1基因扩增产物经DNA探针分子杂交后的阳性磁珠和阴性磁珠的荧光信号强度。结果表明:从400μl全血中提取了基因组DNA,288份样本的DNA产量平均为3.217±0.715μg,A260/A280值平均为1.710±0.103,A260-A320/A280-A320值平均为1.761±0.151;用琼脂糖凝胶电泳法测得DNA的分子量均大于15kb;每一样本HLA-A、-B和-DRB1杂交后的阳性磁珠的荧光信号强度均>600RFU,而阴性磁珠的荧光信号强度<50RFU。结论:本方法适用于从大量全血样本中快速提取基因组DNA,所得的基因组DNA适用于高通量中华骨髓捐献者样本的HLA流式磁珠基因分型等下游的移植免疫学与分子生物学实验。This study was aimed to develop and establish an efficient method for high through-put automatically extracting genornic DNA from EDTA-anticoagulated whole blood samples, and to utilize this method in routine rSSO HLA genotyping by luminex flow array assay, the genomic DNA was extracted automatically from 400 03 blood samples by using TECAN DNA workstation and 96-well plate with 2 ml volume per well. The yield and purity of each DNA sample was tested by UV-spectrophotometer, the integrality of these DNA samples were run electrophoresis on the agarose gel. Each DNA sample was subjected to PCR amplification and hybridization using One lambada rSSO HLA-A, -B and -DRB1 commercial kit, the fluorescent intensity for positive bead and negative bead hybridized with HLA-A, -B and -DRB1 PCR products were calculated and analyzed. The results showed that the mean yield and purity（ A260/A280 ） of genomic DNA extracted from 400 μl whole blood samples were 3. 217 ±0.715 μg and 1. 710 ±0. 103 respectively. The molecular weight was more than 15 kb in size and the fluorescent intensity for positive bead hybridized with HLA-A, -B and -DRB1 PCR products of each sample was 〉 600 RFU, however, the fluorescent intensity for negative bead for each sample was 〈 50 RFU. It is concluded that the highly qualified genomic DNA can be extracted automatically from blood samples of marrow-donors by using TECAN DNA workstation, and the extracted DNA samples are suitable for high through-put HLA genotyping by luminex flow array assay and other downstream transplant immunological and molecular biological experiments.

关 键 词：基因组DNA 基因组DNA高通量提取 中华骨髓库 HLA基因分型 

分 类 号：Q503[生物学—生物化学]