摘要
目的探讨磁珠法DNA提取技术在多重连接依赖探针扩增(MLPA)检测样品制备中的应用价值。方法分别用半自动磁珠法和手工离心柱法提取1例21-羟化酶缺乏症患者和16例健康男性外周血、16例羊水细胞标本的基因组DNA,用MLPA法检测CYP21A2基因拷贝数,比较两种方法制备DNA的质量及MLPA结果。用磁珠法提取200例羊水细胞DNA,MLPA法检测染色体非整倍体异常表达情况。结果磁珠法提取外周血DNA的浓度和总量均高于离心柱法(P<0.05)。两种方法提取羊水DNA浓度相当,磁珠法所得总量较高(P<0.05)。21-羟化酶缺乏症患者CYP21A2基因拷贝数比值接近0.5。200例羊水标本中检出正常二倍体188例,21三体9例,18三体2例,特纳(Turner)综合征(45,X)1例,与核型分析结果一致。结论磁珠法制备的外周血和羊水细胞DNA适用于MLPA检测,具有较高的应用价值。
Objective：To investigate the application of magnetic bead-based DNA extraction technique for the sample preparation of multiplex ligation-dependent probe amplification （MLPA） analysis. Methods：Genomic DNA of peripheral blood samples from 16 healthy male volunteers and a patient with 21-hydroxylase deficiency, as well as amniotic fluid cells from 16 pregnant women was extracted by semiautomatic magnetic bead based DNA extraction method and manual spin column based DNA extraction method. The concentration and purity of DNA samples were compared. The copy numbers of CYP21A2 gene in these samples were determined by MLPA and evaluated. Genomic DNA of amniotic fluid cells from 200 pregnant women was extracted by magnetic bead-based DNA extraction method and the abnormal expressions of chromosomal aneuploidies in the samples were detected by MLPA. Results：Both the concentrations and amounts of genomic DNA extracted from peripheral blood samples by magnetic bead-based DNA extraction were higher than those extracted by spin column based DNA extraction（P〈0.05）, while the concentrations of DNA extracted from amniotic fluid cells by magnetic bead based method were similar to those extracted by spin column based method. The amounts of genomic DNA extracted from amniotic fluid cells by magnetic bead based DNA extraction method were higher than those extracted by spin column based DNA extraction （P〈0.05）. The ratio value of copy numbers of CYP21A2 gene in the patient with 21 hydroxylase deficiency was close to 1, which was similar in both extraction by magnetic bead based and spin column based method. Among the 200 samples of amniotic fluid cells, 9 cases of trisomy 21, 2 cases of trisomy 18 and a case of Turner syndrome （45, X） were identified by MLPA, and the other 188 cases were normal diploid. All of these results were concordant with those of karyotyping analysis. Conclusion：The magnetic bead-based DNA extraction method could be used to prepare cellular DNA samples from peripheral blood and amniotic fluid for MLPA analysis.
出处
《临床检验杂志》
CAS
CSCD
北大核心
2014年第2期86-89,共4页
Chinese Journal of Clinical Laboratory Science
基金
江苏省医学创新团队与领军人才课题(LJ201109)
江苏省临床医学科技专项(BL2012039)
江苏省妇幼保健科研项目(F201206)
南京市科技发展计划项目(201201064)
南京市卫生局科技发展项目(YKK11059)
关键词
磁珠法
DNA提取
多重连接依赖探针扩增技术
magnetic bead
DNA extraction
multiplex ligation-dependent probe amplification
