Improved screening of cDNAs generated by mRNA differential display enables the selection of true positives and the isolation of weakly expressed messages

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 o...

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Bibliographic Details
Published in:Plant molecular biology reporter 1997-09, Vol.15 (3), p.236-245
Main Authors: Zegzouti, H. (UA-INRA, Toulouse, France.), Marty, C, Jones, B, Bouquin, T, Latche, A, Pech, J.C, Bouzayen, M
Format: Article
Language:English
Subjects:
Abundance
ADN
Agricultural sciences
Agronomy
AMELIORATION DES PLANTES
ARN MENSAJERO
ARN MESSAGER
Biotechnology
CLONACION
CLONAGE
CLONING
COMPLEMENTARY DNA
Deoxyribonucleic acid
Differential display
DNA
DNA sequencing
ENSAYO
EXPRESION GENICA
EXPRESSION DES GENES
FITOMEJORAMIENTO
GENE EXPRESSION
Life Sciences
Light emitting diodes
LYCOPERSICON ESCULENTUM
MESSENGER RNA
METHODE
METHODOLOGY
METHODS
METODOS
mRNA
PLANT BREEDING
Polymerase chain reaction
Screening
SELECCION
SELECTION
TESTAGE
TESTING
Tomatoes
Vegetal Biology
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Description
Summary: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.
ISSN:0735-9640
1572-9818
DOI:10.1023/a:1007482318668