Mapping of gene transcripts by nuclease protection assays and cDNA primer extension.
Mapping of gene transcripts by nuclease protection assays and cDNA primer extension.
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DOI：
10.1016/0076-6879(87)52069-9
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发表时间：
1987
影响因子：
--
通讯作者：
F. Calzone;R. Britten;E. Davidson
中科院分区：
文献类型：
--
作者：
F. Calzone;R. Britten;E. Davidson
在基因的分子表征中经常面临的一个重要问题是转录成RNA的那些基因组序列的精确映射。这需要鉴定启动基因转录的基因组位点、在RNA加工期间从初级基因转录物中去除的基因组序列的位置以及终止加工的基因转录物的序列的知识。这里描述的协议的目的是利用相对未表征的基因片段的转录图谱的生成。基本方法是单链DNA探针与细胞RNA杂交，然后用不攻击DNA-RNA杂交体的单链特异性核酸酶处理，以破坏任何未反应的探针序列。因此，杂合双链体中包含的探针序列被保护免于核酸酶消化。通过凝胶电泳确定的受保护的探针片段的大小对应于杂交的序列元件的长度。
An important problem often faced in the molecular characterization of genes is the precise mapping of those genomic sequences transcribed into RNA. This requires identification of the genomic site initiating gene transcription, the location of genomic sequences removed from the primary gene transcript during RNA processing, and knowledge of sequences terminating the processed gene transcript. The objective of the protocols described here is the generation of transcription maps utilizing relatively uncharacterized gene fragments. The basic approach is hybridization of a single-stranded DNA probe with cellular RNA, followed by treatment with a single-strand-specific nuclease that does not attack DNA-RNA hybrids, in order to destroy any unreacted probe sequences. Thus the probe sequences included in the hybrid duplexes are protected from nuclease digestion. The sizes of the protected probe fragments determined by gel electrophoresis correspond to the lengths of the hybridized sequence elements.