摘要
目的:制备NIDD基因TaqM an探针建立检测NIDD基因荧光定量PCR(FQ-PCR),以进一步研究NIDD的表达情况。方法:采用RT-PCR法,从出生后一天的SD大鼠脑组织mRNA中扩增NIDD基因的部分编码序列区(CDS)片段,克隆入pGEM-T载体,筛选阳性克隆、酶切鉴定及序列测定。采用TaqM an探针,以重组质粒为模板绘制标准曲线。结果:RT-PCR法扩增出一特异产物与预期长度112 bp相符,DNA序列测序的结果与GeneBank提供的已知序列(AB098078)比较,所克隆的NIDD基因片段与其中的112 bp完全相同,与NIDD基因100%同源。以重组质粒为模板绘制标准曲线,相关系数r大于0.99,反应效率显示为1,各点变异系数小于10%。结论:采用RT-PCR和T载体技术成功制备出可应用于FQ-PCR的NIDD基因TaqM an探针。
Objective： To obtain the probe of a novel nNOS-interacting DHHC domain-containing protein with dendritic mRNA （NIDD） for real-time fluorescent quantitative PCR （FQ-PCR）. Methods： The partial CDS of NIDD was amplified by RT-PCR from ld postnatal SD rat brain. RT-PCR product was ligated into pGEM-T vector, and the DNA sequence was detected. The standard curve was drawn with the recombinant plasmids and the NIDD TaqMan probe. Results： The product of RT-PCR was 112 bp which matched the size of the purpose. This DNA sequence was 100% homogeneous with the NIDD cDNA reported. The correlation coefficient of the standard curve was above 0.99, the reaction efficiency was 1 and all the variation coefficients were below 10%. Conclusion： The NIDD TaqMan probe for FQ-PCR has been successfully obtained with RT-PCR and T vector techniques.
出处
《江苏大学学报（医学版）》
CAS
2006年第3期185-188,共4页
Journal of Jiangsu University：Medicine Edition
基金
国家自然科学基金资助项目(30300099)
江苏省自然科学基金(BK2003035)
江苏省高校自然科学研究基金(03KJB180109)
江苏省社会发展科技指导性计划项目(BS2004526)
