摘要
目的研究和建立从大量全血标本中提取基因组DNA的有效方法,以应用于Luminex HLA流式磁珠基因分型。方法使用自动工作站(瑞士TECAN公司)从600μl全血中提取基因组DNA,提取的DNA样本用紫外分光光度仪测定其浓度和纯度,DNA的完整性用琼脂糖凝胶电泳检测,并统计分析每一DNA样本流式磁珠HLA-A,B和DRB1基因扩增产物经探针分子杂交后的荧光信号强度。结果从600μl全血中提取基因组DNA,含量平均为10.052±0.824μg,样本的A260nm/A280nm值平均为1.821±0.201。琼脂糖电泳法测得DNA的分子量约为21kb,HLA-A,B和DRB1位点PCR产物杂交后的平均荧光信号强度分别为2 183.84±478.12(exon2),2 168.28±338.59(exon3);2 057.27±397.41(exon2),1959.78±283.24(exon3);3 643.38±327.85。结论此方法适用于从大量全血样本中快速提取基因组DNA,所得基因组DNA适用于高通量HLA流式磁珠基因分型等下游的分子生物学实验。
Objective To develop and establish a effective method for high-volume automatically extracting genomic DNA from whole blood samples,and utilize this method in HLA genotyping by luminex flow array assay. Methods Genomic DNA was extracted automatically from 600/11 blood samples by TECAN DNA workstation. The yield and purity of each sample DNA was tested by UV-spectrophotometer,the integrality of these DNA samples were run electrophoresis by agarose gel, and the fluorescent intensity of HLA-A, B and DRB1 PCR products hybridized with specific probes were calculated and analyzed. Results The mean yield and purity（A260nm/A280nm）of genomic DNA extracted from whole blood samples were 10. 052±0. 824μg and 1. 821±0. 201 respectively, the molecular weight was approximate 21k5. The fluorescent intensity for HLA-A, B and DRB1 PCR products hybridized with specific probes were 2 183.84±478.12（exon2）,2 168.28 ± 338.59 （exon3） ;2 057.27±397.41 （exon2）, 1 959.78±283.24 （exon3）and 3 643.38 ± 327.85 respectively. Conclusion This highly qualitied genomic DNA can be extracted automatically from whole blood samples by using TECAN DNA workstation, and the isolated DNA samples were suitable for HLA genotyping by luminex flow array assay and other downstream molecular biological experiments.
出处
《现代检验医学杂志》
CAS
2007年第3期1-3,共3页
Journal of Modern Laboratory Medicine
基金
深圳市医疗卫生系统2005年科技计划项目:无关供/受者HLA-C基因测序配型及其临床效果(2005年序号169)
