Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True... The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative low abundance differential clones. Following cloning, a second reverse Northern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very low abundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Molecular Biology Reporter Springer Journals

Improved Screening of cDNAs Generated by mRNA Differential Display Enables the Selection of True Positives and the Isolation of Weakly Expressed Messages

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Publisher
Springer Journals
Copyright
Copyright © 1997 by Kluwer Academic Publishers
Subject
Life Sciences; Plant Sciences; Plant Physiology
ISSN
0735-9640
eISSN
1572-9818
D.O.I.
10.1023/A:1007482318668
Publisher site
See Article on Publisher Site

Abstract

The high percentage of false positives generated by differential display (as high as 85%) has previously limited the potential of the method. This report describes an efficient methodology that enables false positives to be discarded prior to cloning, via reverse Northern analysis. This first step of the screening also allows the detection of putative low abundance differential clones. Following cloning, a second reverse Northern combined with partial DNA sequencing and RT-PCR detection allows isolation of all differential cDNAs including very low abundance clones. Use of the sequential screening procedure described here led to the isolation of novel tomato genes responding to the plant hormone ethylene while minimising labor and materials input.

Journal

Plant Molecular Biology ReporterSpringer Journals

Published: Sep 29, 2004

References

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