适配器(计算)
互补DNA
逆转录酶
cDNA文库
cDNA末端的快速扩增
底漆(化妆品)
生物
分子生物学
基因
计算生物学
化学
遗传学
分子克隆
计算机科学
核糖核酸
有机化学
操作系统
作者
Maarten Kooiker,Gang‐Ping Xue
出处
期刊:Methods in molecular biology
日期:2013-10-31
卷期号:: 29-40
被引量:6
标识
DOI:10.1007/978-1-62703-715-0_5
摘要
The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5′ and 3′ end of the cDNA. The 3′ adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5′ adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.
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