A primer extension method for quantitation of in vitro synthesized mRNA transcripts directed by promoter elements containing multiple transcription initiation sites.
A primer extension method for quantitation of in vitro synthesized mRNA transcripts directed by promoter elements containing multiple transcription initiation sites.
复制标题
一种引物延伸方法，用于对由含有多个转录起始位点的启动子元件引导的体外合成的 mRNA 转录物进行定量。
DOI：
10.1006/abio.1995.0018
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Johannes,G
中科院分区：
文献类型：
--
作者：
Kosovsky,MJ;Johannes,G
材料和方法本方法用于检测质粒和荧光素酶的水平。来自SLuc2和SLuc2的质粒转导活性含有位于Up-SV2a的SV40Ep(无增强子)，这是一个单一的延伸产物，带有预测的荧光素酶报告基因流(13)。观察到SV2a的长度(图2a)。荧光素酶延伸产物上游含有SV40Ep/增强子的存在促进了定量基因的表达(11)。荧光素酶引物包括对荧光素酶转录本的24个碱基的分析。转录寡核苷酸，5-GGGCCTTTCTTATGTTTTTGTTTTGG-来自0.3和0.6毫克SV2A的活性是CG-3，用[g-32P]ATP末端标记(分别由2.4和3.4倍的Ci/mmo1诱导7000，ICN制药公司)。标记的SLuc2(通道4-7)观察到。然后用SV40增强子IS CON-8M尿素凝胶从20%的聚丙烯酰胺/转录活性中纯化出诱导引物。与以前的工作一致(11)。此外，由于与ddGTP的DNA链终止具有一致性-
MATERIALS AND METHODSWhen this method was used to examine levels of Plasmids and luciferase primer. The plasmid transciptional activity derived from SLuc2 and SLuc2 contains the SV40Ep (enhancerless) located up-SV2A, a single extension product with the predicted stream of the luciferase reporter gene (13). SV2A con-length was observed (Fig. 2A). The presence of a sintains the SV40Ep/enhancer upstream of the luciferase gle extension product facilitated the quantitative gene (11). The luciferase primer consisted of a 24-base analysis of luciferase transcripts. Transcriptional oligonucleotide, 5-GGGCCTTTCTTTATGTTTTTGG-activity derived from 0.3 and 0.6 mg of SV2A was CG-3, that was end-labeled using [g-32P] ATP (7000 induced by 2.4-and 3.4-fold, respectively, over that Ci/mmol, ICN Pharmaceuticals, Inc.). The labeled observed with SLuc2 (lanes 4–7). The induction of primer was then purified from a 20% polyacrylamide/transcriptional activity by the SV40 enhancer is con-8 M urea gel. sistent with previous work (11). Furthermore, since DNA chain termination with ddGTP has consoli-