Entwicklung einer auf subtraktiver Hybridisierung basierenden Nachweismethode zur Identifizierung differentieller Genexpression in Eukaryonten

Abstract

A routine suitable genome subtraction system for the analysis of differential gene expression in high complex eucaryotes is constructed. This process is robust, relative sensitive, with low costs and non-radioactive. It represents a good alternative to other known methods. The development of this method is based on SSH and DSC.First it was found out that with SSH and 2D-Gel, it is not able to isolate differential expressed genes in potatoes. Although the reporter target is enriched by SSH, the fragment with highest concentration in the SSHG/D-products is not from the target, but from one of the high concentrated background sequences.It was then proven that a subtraction by DSC is not possible at all. A hypothesis on the non-functioning of DSC was set up and proven to be true: the tester-overhangs with 24 bp are so long that, under the functioning buffer and temperatur conditions for MBN, they build up dsDNAs which can not be digested by MBN in a ssDNA specific fashion. An efficient subtraction is therefore not possible.Based on the above findings an efficient genome-subtraction system called NSC was developed, which differ from DSC by its innovative adapters/primers. An efficient subtraction achieved by NSC was demonstrated on a lambda fragment.Subsequently SNH (subtractive, negative hybridization) was established, which combines the sensitivity of SSH-detection and the efficiency of the NSC-subtraction. It is shown on the example of potato cDNAs that SNH is able to directly isolate differential gene expression in high eucaryots. The sensitivity of SSH is figured out to be 500 ppm foreign fragment in potato cDNAs, that of SNC (under conditions tested) to be 100 ppm instead. For the detection of differentially expressed genes in eucaryotes, this increase in sensitivity marks an increase in quality, because SNH is capable to directly (without screening) identify cDNAs in the middle concentrated class, to whom most of sequences responsible for differentiation belong.