Increased specificity of reverse transcription griming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display
Increased specificity of reverse transcription griming by trehalose and oligo-blockers allows high-efficiency window separation of mRNA display
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DOI：
10.1093/nar/27.5.1345
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发表时间：
1999-03-01
影响因子：
14.9
通讯作者：
Hayashizaki, Y
中科院分区：
文献类型：
--
作者：
Mizuno, Y;Carninci, P;Hayashizaki, Y
我们已经开发了一种方法，通过提高特异性的启动在逆转录中的cDNA展示的高效率的窗口分离。在常规方法中，使用两个碱基锚定的寡（dT）引物（5 ′ dT（16）VN3 ′，其中N是任何碱基，V是G、A或C）来制作用于展示转录物的窗口。然而，逆转录酶经常延伸错误引发的寡核苷酸。为了避免dT（16）VN引物的错误引发，我们开发了两种新技术。一种是用二糖海藻糖热激活的逆转录酶进行高温引发。另一种是使用与非选择性引发的mRNA杂交的竞争性寡核苷酸阻断剂，防止VN位点的错误引发。这些方法相结合，以改善限制性标志cDNA扫描（RLCS），从而消除出现在不同的窗口中的冗余信号。这是通过从poly（A）位点开始增加逆转录起始的特异性来实现的。这种方法为转录本的精确可视化铺平了道路，从而可以理解个体组织和每个发育阶段的表达谱。
We have developed a method for high-efficiency window separation of cDNA display by increasing the specificity of priming in reverse transcription. In the conventional method, two-base anchored oligo(dT) primers (5'dT(16)VN3', where N is any base and V is G, A or C) are used to make windows for the display of transcripts. However, reverse transcriptase often extends misprimed oligonucleotides. To avoid mispriming from dT(16)VN primers, we have developed two new technologies. One is higher temperature priming with reverse transcriptase thermoactivated by the disaccharide trehalose. The other is the use of competitive oligonucleotide blockers that hybridize to the non-selectively primed mRNAs, preventing the mispriming from the VN site. These methods were combined to improve restriction landmark cDNA scanning (RLCS), resulting in the elimination of the redundant signals that appear in different windows. This was achieved by the increased specificity of initiation of reverse trans-cription from the beginning of poly(A) sites. This method paves the way for the precise visualization of transcripts to allow expression profiles in individual tissues and at each developmental stage to be understood.