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介绍一种从不同类型细胞或不同生长状态细胞中分离差异表达基因的快速高效mRNA差异显示技术 ,其特点是利用Ready To GoRT PCR反应珠和Ready To GoRAPD分析珠进行mRNA差异显示分析 ,使取样步骤降至最低程度 ,减少了潜在的取样误差和外源DNA污染 ,并确保每次反应的高度重复性 .通过银染测序胶分析差异显示的cDNA带 ,便于DNA回收和进一步克隆 .用此方法分析人胚发育早期不同阶段基因的差异表达 ,选用6条随机引物对 3、 4和 5周龄人胚进行mRNA差异显示分析 ,从 2 0 0 0多条带中共分离出 14个差异产物 ,经二次扩增及反向RNA印迹确证其中 6个片段为发育不同阶段差异表达基因
A rapid and efficient mRNA differential display technique for separating differentially expressed genes from different types of cells or cells of different growth states is characterized in that mRNA differential display analysis is performed using Ready To GoRT PCR reaction beads and Ready To GoRAPD analysis beads so that the sampling step Minimize potential sampling errors and exogenous DNA contamination and ensure a high degree of reproducibility for each reaction Differentially displayed cDNA bands are analyzed by silver-stained sequencing gel for DNA recovery and further cloning Analysis by this method The differential expression of genes in different stages of early human embryo development was analyzed. Six different random primers were used to analyze the mRNA differences of three, four and five weeks old human embryos. 14 differential products were isolated from more than 200 bands. Sub-amplification and reverse Northern blot confirmed that six of the fragments were differentially expressed in different stages of development