纸基法血液基因组DNA的提取与应用

Extraction and Application of Blood Genomic DNA by Paper-based Method

建立一种提取小鼠血液中基因组DNA的纸基方法进行分子鉴定和基因分型,选择试剂盒提取法和常规煮沸法作为对比,分别对10只基因敲除型小鼠后代组织的基因组DNA进行提取,用多基因PCR扩增进行鉴定,随机选择2只小鼠的鉴定结果进行测序验证。PCR均扩增出符合预期大小的条带,验证了纸基法的稳定性和特异性。3种DNA提取方法的PCR扩增结果表明,试剂盒提取法与纸基法提取效果相似,共鉴定出4只野生型小鼠,5只基因敲除杂合型小鼠和1只基因敲除型纯合型小鼠;煮沸法对小鼠基因型DNA提取效果较差。测序分析结果显示,基因类型与纸基法鉴定基因类型一致。用纸基法提取基因组DNA不需要专业设备就可满足小鼠血液基因组DNA的提取及下游试验,可为基因型鉴定提供一种可靠快速的方法。

A simple and rapid paper-based method for extracting genomic DNA from mouse blood was established by Whatman No.1 cellulose filter paper,lysate and eluent,and molecular identification and genotyping were carried out by this method.In this experiment,the kit extraction method and the conventional boiling method were selected as the comparison.The genomic DNA of the offspring tissues of 10 gene knockout mice was extracted and identified by multi-gene PCR amplification.The identification results of 2 mice were randomly selected for sequencing verification.PCR amplified bands in line with the expected size,which verified the stability and specificity of the paper-based method.The PCR amplification results of three DNA extraction methods showed that the kit extraction method was similar to the paper-based method.A total of 4 wild-type mice,5 gene knockout heterozygous mice and 1 gene knockout homozygous mice were identified.The boiling method had a poor effect on the extraction of mouse genotype DNA.The results of sequencing analysis showed that the gene type was consistent with the gene type identified by paper-based method.In this study,the paper-based method was used to extract genomic DNA without professional equipment to meet the extraction of genomic DNA from mouse blood and downstream experiments,providing a reliable and rapid method for genotype identification and measurement.