Gastel J A, Sutter T R
Div. of Toxicological Sciences, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD 21205-2179, USA.
Biotechniques. 1996 May;20(5):870-5. doi: 10.2144/96205st07.
Most cDNA enrichment techniques involve technically complicated subtractive and hybridization reactions whose kinetics have not been characterized. We describe a control for cDNA enrichment procedures based on commercially available reagents, and we use this control to optimize a commonly used subtractive hybridization reaction. Using this control, we show that high-abundance transcripts can be efficiently removed following very short hybridization reactions. With the addition of unlabeled rat liver cDNA, we develop a system that mimics the reaction kinetics of complex cDNA pools. Because this system enables the measurement of specific vs. nonspecific hybridization and subtraction in the same tube and is an effective control for all steps in the cDNA enrichment procedure, it facilitates the development and optimization of novel cloning techniques for a desired abundance level of differentially expressed cDNAs.
大多数cDNA富集技术涉及技术复杂的消减和杂交反应，其动力学尚未得到表征。我们描述了一种基于市售试剂的cDNA富集程序对照，并使用该对照优化常用的消减杂交反应。使用该对照，我们表明在非常短的杂交反应后，高丰度转录本可以被有效去除。通过添加未标记的大鼠肝脏cDNA，我们开发了一个模拟复杂cDNA池反应动力学的系统。由于该系统能够在同一管中测量特异性与非特异性杂交及消减，并且是cDNA富集程序所有步骤的有效对照，它有助于开发和优化针对所需丰度水平的差异表达cDNA的新型克隆技术。
