Konietzko U, Kuhl D
Zentrum für Molekulare Neurobiologie (ZMNH), University of Hamburg, Martinistrabetae 52, D-20246 Hamburg, Germany.
Nucleic Acids Res. 1998 Mar 1;26(5):1359-61. doi: 10.1093/nar/26.5.1359.
Moderately induced genes often escape detection in conventional subtraction hybridisation cloning. Here a modification of a phagemid subtraction protocol is described that overcomes this problem. The protocol uses low ratio hybridisation of driver to target sequences to allow enrichment of the sequences of interest, and back-hybridisation of the subtracted sequences with induced sequences to reduce the accumulation of false positive clones. The procedure takes advantage of the quantitative representation of cellular RNA populations in cDNA libraries, therefore, they may serve not only as renewable sources of driver and target sequences, but also as sources of population cRNAs used in northern blots and differential Southern blots.
中度诱导的基因在传统的消减杂交克隆中常常难以被检测到。本文描述了一种噬菌粒消减方案的改进方法，该方法克服了这一问题。该方案利用驱动序列与靶序列的低比例杂交来富集感兴趣的序列，并使消减后的序列与诱导序列进行反向杂交，以减少假阳性克隆的积累。该方法利用了cDNA文库中细胞RNA群体的定量表示，因此，它们不仅可以作为驱动序列和靶序列的可再生来源，还可以作为用于Northern印迹和差异Southern印迹的群体cRNA的来源。
