矩阵设计检测金针菇转化子外源DNA插入位点

插入位点分析对于金针菇功能基因组学的研究极为重要,分析方法常用反向PCR、热不对称交错PCR、Tail-PCR、染色体步移等,存在操作复杂、消耗时间长、特异性较差、效率低等缺点。近年来开始应用基因组重测序的方法,对转化子逐一测序与分析,工作量较大、费用较高。本研究应用矩阵设计,把多个转化子的DNA混合构成样品池,重测序后分析插入位点,M个样品池的测序数据可分析M×(M+1)/2个转化子的插入位点。应用矩阵设计构建6个样品池检测21个转化子,获得21个插入位点,表明这种方法可行、适合大样本分析,如突变体库。

Identification of exogenous DNA insertion site of transformants in Flammulina filiformis using matrix design

Insertion site analysis is very important in functional genomics research of Flammulina filiformis. The analysis methods commonly used are reverse PCR, thermal asymmetric interlaced PCR, Tail-PCR, chromosome walking, etc., and these methods are hard to operate, time-consuming, poorly specific and inefficient. In recent years, the next generation sequencing has been applied in identification insertion site that transformants were sequenced one by one, but it has a large workload and a high cost. In this study, a matrix design was used to mix the DNA of multiple transformants to form a sample pool. After resequencing, the insertion sites were analyzed. The sequencing data of M sample pools can analyze the insertion sites of M× (M+1)/2 transformants. The matrix design was used to detect 21 transformants, and 21 insertion sites were identified. This method indicates that matrix design is practicable for insertion site identification and suitable for large sample analysis, such as mutant library.