Rapid and sensitive analysis of mRNA polyadenylation states by PCR.
Rapid and sensitive analysis of mRNA polyadenylation states by PCR.
复制标题
DOI：
10.1101/gr.4.6.317
复制
发表时间：
1995-06
期刊：
影响因子：
--
通讯作者：
F. Salles;S. Strickland
中科院分区：
文献类型：
--
作者：
F. Salles;S. Strickland
本文描述了一种快速灵敏的技术，可以在亚纳克细胞总RNA的范围内测量特定mRNA上的poly(A)尾巴的长度[poly(A) test (PAT)]。在单管反应中，利用寡核苷酸（dT）和DNA连接酶在mrna的聚(a)尾部原位合成了一个聚（dT）引物。通过调节退火温度和引物浓度，一个富含gc的适配器序列被定位到poly（dT）引物的5'端。这个连接的聚（dT）锚随后被用来启动mRNA的逆转录，产生一个PAT cdna文库。poly(a) tail的长度是通过使用oligo（dT）锚定引物和特定信息引物的PCR扩增来确定的。比较不同样品的PCR产物可以定量测定给定mRNA的聚腺苷化变化。该技术克服了许多与传统聚(A)尾部长度评估相关的缺陷，并应证明在研究与聚腺苷化有关的各种过程中是有用的。
A rapid and sensitive technique is described that measures the length of the poly(A) tail on a specific mRNA within subnanogram quantities of total cellular RNA [the Poly(A) test (PAT)]. In a single-tube reaction, a poly(dT) primer is synthesized in situ on the poly(A) tail of mRNAs using oligo(dT) and DNA ligase. By modulating the annealing temperature and primer concentrations, a GC-rich adapter sequence is targeted to the 5' end of the poly(dT) primer. This ligated poly(dT)-anchor is then used to prime reverse transcription of the mRNA, yielding a library of PAT cDNAs. The length of a poly(A) tail is determined by PCR amplification using the oligo(dT)-anchor primer and a message-specific primer. Comparison of PCR products from different samples allows quantitative determination of changes in polyadenylation of a given mRNA. This technique overcomes many of the pitfalls associated with conventional poly(A) tail length assessments and should prove useful in studying a variety of processes relating to polyadenylation.