Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.
Terminal transferase-dependent PCR: a versatile and sensitive method for in vivo footprinting and detection of DNA adducts.
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末端转移酶依赖性 PCR：一种用于体内足迹和 DNA 加合物检测的通用且灵敏的方法。
DOI：
10.1093/nar/26.7.1807
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发表时间：
1998
影响因子：
14.9
通讯作者：
Riggs,AD
中科院分区：
文献类型：
--
作者：
Komura,J;Riggs,AD
我们报道了一种新的、灵敏和通用的基因组测序方法，该方法可用于活体足迹和DNA加合物的研究。从哺乳动物基因组DNA开始，单链产物通过重复的引物延伸得到；这些产物经过末端脱氧核苷酸转移酶在3‘端的同聚核苷酸拖尾，然后连接到具有互补的3’端的双链接头，并用于聚合酶链式反应。这种末端转移酶依赖的聚合酶链式反应(TDPCR)方法可以产生比传统的连接介导的聚合酶链式反应(LMPCR)强许多倍的条带信号。利用TDPCR可以很容易地检测到小鼠Xist基因启动子中的紫外光足迹。将DNA加合物转化为链断裂不需要特殊的酶或化学试剂。任何阻碍引物延伸的损伤都应该是可以检测到的。
We report here a new, sensitive and versatile genomic sequencing method, which can be used forin vivofootprinting and studies of DNA adducts. Starting with mammalian genomic DNA, single-stranded products are made by repeated primer extension; these products are subjected to homopolymeric ribonucleotide tailing at the 3′ termini with terminal deoxynucleotidyl transferase and then ligated to a double-stranded linker having a complementary 3′ overhang, and used for PCR. This terminal transferase-dependent PCR (TDPCR) method can generate band signals many-fold stronger than conventional ligation-mediated PCR (LMPCR). A UV photofootprint in the mouse Xist gene promoter can be easily detected using TDPCR. No special enzymes or chemical reagents are needed to convert DNA adducts into strand breaks. Any lesion that blocks primer extension should be detectable.