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. 2000 Oct 15;28(20):E90.
doi: 10.1093/nar/28.20.e90.

Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization

D V Rebrikov  1 , O V Britanova, N G Gurskaya, K A Lukyanov, V S Tarabykin, S A Lukyanov
Affiliations

Mirror orientation selection (MOS): a method for eliminating false positive clones from libraries generated by suppression subtractive hybridization

D V Rebrikov et al. Nucleic Acids Res. .

Abstract

Suppression subtractive hybridization (SSH) is one of the most powerful and popular methods for isolating differentially expressed transcripts. However, SSH-generated libraries typically contain some background clones representing non-differentially expressed transcripts. To overcome this problem we developed a simple procedure that substantially decreases the number of background clones. This method is based on the following difference between target and background cDNAs: each kind of background molecule has only one orientation with respect to the two different flanking adapter sequences used in SSH, while truly differentially expressed target cDNA fragments are represented by both sequence orientations. The described method selects the molecules that arose due to hybridization of such mirror-orientated molecules. The efficiency of this method was demonstrated in both model and real experimental subtractions.

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Figures

Figure 1
Figure 1
Schematic representation of the MOS method. Rectangles represent DNA molecules (broad rectangles, double-stranded DNA; narrow rectangles, single-stranded DNA). Yellow rectangles, adapter A; green rectangles, adapter B. Pink molecules, target cDNA; blue, background cDNA. The gradient of filling shows the orientation of the cDNA molecules.
Figure 2
Figure 2
Application of the MOS technique to the model subtraction (gel-electrophoresis of the PCR products). Lane M, 1 kb ladder (Gibco BRL). Lanes 1–3, results of applying of SSH and MOS procedures to tester cDNA containing 0.01% ϕX174 DNA; lanes 4–6, tester cDNA containing 0.001% ϕX174 DNA. Lanes 1 and 3, samples after SSH. Lanes 2 and 5, samples after MOS. Lanes 3 and 6, samples after MOS with addition of excess driver. Lane 7, amplification product of ϕX174 DNA digested with HaeIII and ligated with adapters A and B. Small divergences in length of the fragments presenting in SSH-generated and control ϕX174/HaeIII samples on the one hand and in MOS-generated samples on the other hand are due to the different PCR primers used.
See this image and copyright information in PMC

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