DNA工作站在HLA测序分型中的应用

目的 建立从全血样本中高通量提取基因组DNA的方法,以应用于HLA常规测序分型.方法 采用DNA工作站及2 ml深孔板,从400份全血样本中提取基因组DNA.用紫外分光光度仪测定其浓度和A<,260>/A<,280>值,并采用琼脂糖电泳检测DNA的完整性.扩增产物经纯化后作为测序模板,产物经酒精/EDTA/NaOAc法纯化,用ABI Prism<'TM>3730测序仪电泳检测.相关的分析软件分析HLA基因型.结果 使用400μL全血中400份样本的DNA产量平均为(3.217±0.715)μg,A<,260>/A<,280>值平均为1.710±0.103;琼脂糖电泳表明DNA的分子量均大于15×10<'3>;HLA-A、HLA-B和HLA-DRB1座位序列碱基峰高均在2000以上,测序评分在75分以上.48份HLA-A、B和DRB1基因型已知的室内质控样本,经本文的方法提取DNA并进行HLA基因分型,经盲检质控检测,所得到的结果与已知的HLA基因型相一致.结论 采用本方法所提取的DNA能稳定、可靠地应用于骨髓捐献者样本的HLA-A、B和DRB1座位测序分型,具有快速、高通量化的特点和广泛的应用前景.

DNA work station and its application in HLA sequence-based typing

Objective To develop and establish an efficient method for high throughput automatically extracting genomic DNA from peripheral whole blood samples and utilize this method for HLA sequencing based typing.Methods Genomic DNA was extracted automatically from 400 μL blood samples 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 analyzed by the agarose gel electrophoresis.The purified PCR product was utilized as the DNA template in the sequencing reaction.Sequencing reaction products were purified by EtOH/EDTA/NaAc method and then were electrophoresis by ABI Prism 37300 DNA Sequencer.Results The mean yield and purity(A260/A280) of genomic DNA extracted from 400 μL whole blood samples were(3.2175±0.715) μg and(1.7105±0.103),respectively.The molecular weight of DNA was all more than 15 kb.The apex heights of HLA-A,B and DRB1 sequencing are all above 2000,and BCS are above 75.The HI.A-A,B and DRB1 genotyping results of 48 coded quality control samples were completely in accordance with the genotypes as previously.Conclusion The developed genomic DNA extracting method is suitable for HLA genotyping and can provide reliable genotyping results for samples from unrelated marrow donor samples.This high through-put DNA extracting method showed a broad application in other molecular biological experiments.