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Related Experiment Videos

Representative cDNA libraries from few plant cells

T Dresselhaus1, H Lörz, E Kranz

  • 1Zentrum für Angewandte Molekularbiologie der Pflanzen, AMP II, Universität Hamburg, Germany.

The Plant Journal : for Cell and Molecular Biology
|April 1, 1994
PubMed
Summary

Researchers developed a novel reverse transcriptase/polymerase chain reaction (RT/PCR) method to create complementary DNA (cDNA) libraries from limited plant cells. This technique successfully generated the first cDNA library from maize egg cells, advancing plant molecular biology research.

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Area of Science:

  • Molecular Biology
  • Plant Science
  • Genomics

Background:

  • Construction of complementary DNA (cDNA) libraries is crucial for gene expression studies.
  • Limited cell numbers pose a significant challenge for traditional cDNA library synthesis.

Purpose of the Study:

  • To develop a novel reverse transcriptase/polymerase chain reaction (RT/PCR) method for generating representative cDNA libraries from a small number of isolated cells.
  • To construct the first cDNA library from maize egg cells.

Main Methods:

  • Extraction of poly(A)+ RNA from approximately 100 maize cells.
  • Reverse transcription to synthesize single-stranded cDNA (sscDNA).
  • dG-tailing of sscDNA and amplification via a two-step polymerase chain reaction (PCR).
  • Cloning of amplified cDNA into the lambda Uni-ZAP XR vector.

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Main Results:

  • The RT/PCR method yielded full-size cDNAs, with the majority being less than or equal to 2 kilobase pairs (kbp).
  • A primary library of 6.8 x 10(6) plaque-forming units (p.f.u.) was constructed from 128 maize egg cells, with an average insert size of 860 base pairs (bp).
  • 0.31% of clones hybridized to a cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe, indicating successful library representation.

Conclusions:

  • The described RT/PCR method enables the construction of representative cDNA libraries from very few cells.
  • This study reports the generation of the first cDNA library from higher plant egg cells, providing a valuable resource for future research.