A PRACTICAL APPROACH FOR QUANTITATING SPECIFIC MESSENGER-RNAS BY SOLUTION HYBRIDIZATION
A PRACTICAL APPROACH FOR QUANTITATING SPECIFIC MESSENGER-RNAS BY SOLUTION HYBRIDIZATION
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DOI：
10.1016/0003-2697(83)90188-4
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发表时间：
1983-01-01
影响因子：
2.9
通讯作者：
PALMITER, RD
中科院分区：
文献类型：
--
作者：
DURNAM, DM;PALMITER, RD
本文描述了一种特定的c[互补]DNA探针的制备和使用，用于通过溶液杂交定量mRNA。克隆的DNA序列经过缺口翻译、变性、杂交到含有信息链（mDNA）序列的单链M13克隆中，然后在Bio-Gel A50上进行色谱分离，首先是天然条件，然后是变性条件，得到单链cDNA探针。详细的溶液杂交测定，其中单链cDNA用于定量mRNA在总核酸样品描述。在样品分离后的一天内，可以很容易地检测到0.5 pg的mRNA。该分析既快速又敏感，可用于测量与任何克隆DNA序列互补的RNA。它非常适合于期望对多个样品进行准确定量的情况。
The preparation and use of a specific c[complementary]DNA probe for quantitating mRNA by solution hybridization is described. Cloned DNA sequences are nick translated, denatured, hybridized to single-stranded M13 clones containing message strand (mDNA) sequences, and separated chromatographically on Bio-Gel A50 under first native and then denaturing conditions to yield a single-stranded cDNA probe. The details of a solution hybridization assay in which the single-stranded cDNA is used to quantitate mRNA in total nucleic acid samples are described. As little as 0.5 pg of mRNA can easily be detected within a day of sample isolated. The assay is both rapid and sensitive, and can be used to measure RNA complementary to any cloned DNA sequence. It is ideally suited to situations when accurate quantitation of multiple samples is anticipated.