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Identification of DNA sequences flanking T-DNA insertions by PCR-walking 总被引:13,自引:0,他引:13
Amanda Cottage Aiping Yang Heather Maunders Rosalind C. de Lacy Nicola A. Ramsay 《Plant Molecular Biology Reporter》2001,19(4):321-327
In recent years, concerns over genetic modification issues have resulted in regulatory authorities requiring comprehensive
analysis of transgene insertion events in the plants that are to be commercialized. Determining that plants are devoid of
vector backbone sequences is a trivial task that is best achieved by Southern blot analysis; however, identifying the DNA
sequences flanking the T-DNA insertions can be arduous. In this paper, we present a robust method of characterizing this insertion
event. We have applied and modified a genomic walking method that combines vectorette and suppression PCR walking. 相似文献
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A Johansson BE Engström J Frey K-E Johansson V Båverud 《Acta veterinaria Scandinavica》2005,46(4):241
Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias
in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive
and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic
laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4°C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpb1 and etx genes were detected in all isolates from samples stored either at room temperature or at 4°C for 24–44 h. Repeated aerobic
treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem. 相似文献