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.
Zentrum für Angewandte Molekularbiologie der Pflanzen, AMP II, Universität Hamburg, Germany.
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Lörz
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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