Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7.
Simple rapid method for the synthesis of radioactively labeled cDNA hybridization probes utilizing bacteriophage M13mp7.
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利用噬菌体 M13mp7 合成放射性标记 cDNA 杂交探针的简单快速方法。
DOI：
10.1073/pnas.79.3.724
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发表时间：
1982
影响因子：
11.1
通讯作者：
Kalinyak,JE
中科院分区：
文献类型：
--
作者：
Ricca,GA;Taylor,JM;Kalinyak,JE
将大鼠α1-酸性糖蛋白和大鼠谷胱甘肽S-转移酶mRNA的双链cDNA序列插入噬菌体M13mp7的Pst I位点，并用于开发直接从克隆模板DNA制备特异性cDNA杂交探针的新方法。载体 DNA 中 Pst I 位点周围的回文序列允许从重组噬菌体中分离的单链 DNA 向后折叠，从而形成末端由 M13mp7 单链 DNA 大环和插入的外源 DNA 小环界定的稳定杂交体。将对应于外源DNA内部序列的引物进行杂交，然后使用大肠杆菌DNA聚合酶I合成克隆的插入DNA的32P标记的互补DNA拷贝。通过随后的碱性蔗糖梯度沉淀，很容易分离出单链 cDNA 反应产物。用乙二醛变性后，标记的 cDNA 产物的凝胶电泳表明单个离散条带，其电泳迁移率对应于插入的 DNA 序列的长度。约 95% 的 cDNA 产物在与溶液中过量 RNA 的杂交反应中形成 S1 核酸酶抗性杂交体。在 cDNA 产物中未检测到与 M13mp7 载体 DNA 互补的 DNA 序列。因此，这些 M13mp7 衍生探针的功能相当于 mRNA 的 cDNA 拷贝，可用于 mRNA 浓度的定量测量。这种简单、快速的方法可能可用于大多数克隆的 DNA 序列，以产生单链放射性标记的 DNA，而不会污染载体 DNA 序列，几乎可以满足任何杂交要求。
Double-stranded cDNA sequences for rat alpha 1-acid glycoprotein and rat glutathione S-transferase mRNAs were inserted into the Pst I site of bacteriophage M13mp7 and used to develop a new method for preparing specific cDNA hybridization probes directly from cloned template DNA. A palindrome sequence surrounding the Pst I site in the vector DNA permitted single-stranded DNA isolated from the recombinant phage to fold back, thus forming a stable hybrid bounded on the ends by a large loop of M13mp7 single-stranded DNA and a small loop of inserted foreign DNA. A primer corresponding to an internal sequence of the foreign DNA was hybridized, then Escherichia coli DNA polymerase I was used to synthesize a 32P-labeled complementary DNA copy of the cloned inserted DNA. The single-stranded cDNA reaction product was easily isolated by subsequent sedimentation through alkaline sucrose gradients. Gel electrophoresis of the labeled cDNA product, after denaturation with glyoxal, indicated a single discrete band with an electrophoretic mobility corresponding to the length of the inserted DNA sequence. About 95% of the cDNA product formed S1 nuclease-resistant hybrids in hybridization reactions with excess RNA in solution. DNA sequences complementary to the M13mp7 vector DNA were not detected in the cDNA product. Thus, these M13mp7-derived probes are the functional equivalent of cDNA copies to mRNAs and can be employed for quantitative measurements of mRNA concentration. This simple, rapid method probably can be used for most cloned DNA sequences to yield single-stranded radioactively labeled DNA, without contaminating vector DNA sequences, for virtually any hybridization requirement.