Summary
Transformed root lines of Nicotiana species, containing NPTII and Gus genes, were used to study the parameters affecting the use of the Polymerase Chain Reaction as a routine analytical tool for quickly analysing plant transformants for the presence of a foreign gene. The basic reaction mix as described by Cetus Corporation (Saiki 1989) was close to optimal for successful PCR amplification of internal sequences of both NPTII and Gus from genomic plant DNA. The temperature of primer annealing in the PCR protocols was found to be the most important variable, as low temperatures caused amplification of artefact bands and smearing after analysis on ethidium bromide agarose gels. Various formulae for calculating the Tm for binding of primers of various lengths (20–30 bases) are described in relation to predicting suitable annealing temperatures in the PCR.
For tobacco species the PCR reaction worked efficiently with up to 2 μg of genomic DNA. However, with DNA from Mentha species (mint), an inhibitor of the PCR process was co-extracted with the DNA which prevented amplification of target sequences, if more than 10 ng of genomic DNA was present in the reaction.
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Abbreviations
- c7dCTP:
-
7-deaza-2′-deoxyguanosine
- GUS-β-Glucuronidase, NPT II:
-
neomycin phosphotransferase II
- PCR:
-
polymerase chain reaction
- TBE:
-
Tris Borate
- EtBr:
-
ethidium bromide
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Hamill, J.D., Rounsley, S., Spencer, A. et al. The use of the polymerase chain reaction in plant transformation studies. Plant Cell Reports 10, 221–224 (1991). https://doi.org/10.1007/BF00232562
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DOI: https://doi.org/10.1007/BF00232562