Representative cDNA libraries from few plant cells.
A reverse transcriptase/polymerase chain reaction (RT/PCR) method for the construction of representative cDNA libraries originating from few isolated cells is described. Poly(A)+ RNA was extracted from an average of 100 maize cells and reverse transcribed into sscDNA. The sscDNA was dG-tailed at its 3' end and amplified during a two-step PCR reaction. The generated PCR products were analysed and the majority < or = 2 kbp were full-size cDNAs. A fraction of the amplified cDNA from 128 isolated maize egg cells was cloned into the lambda Uni-ZAP XR vector and a primary library of 6.8 x 10(6) p.f.u. was obtained. The average insert size is 860 bp. It was further determined, that 0.31% of the clones hybridized to a cytosolic GAPDH probe. It is thought that, with this method, the first cDNA library of egg cells in higher plants was generated.
Dresselhaus
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Zentrum für Angewandte Molekularbiologie der Pflanzen, AMP II, Universität Hamburg, Germany.
Lörz
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Kranz
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E
D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction; D011523:Protoplasts; D003313:Zea mays / Q000166:cytology / Q000235:genetics*
D017931:DNA Primers; D018076:DNA, Complementary; D009838:Oligodeoxyribonucleotides; C033538:oligodeoxyguanylic acid