Identification of DNA sequences flanking T-DNA insertions by PCR-walking

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 bes...

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Bibliographic Details
Published in:Plant molecular biology reporter 2001-12, Vol.19 (4), p.321-327
Main Authors: Cottage, A, Yang, A, Maunders, H, de Lacy, R.C, Ramsay, N.A
Format: Article
Language:English
Subjects:
chromosome walking
Commercialization
Deoxyribonucleic acid
DNA
DNA fingerprinting
Gene sequencing
Genetic modification
Insertion
methodology
molecular sequence data
Nucleotide sequence
nucleotide sequences
Polymerase chain reaction
T-DNA
transfer DNA
Transgenic plants
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Summary: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.
ISSN:0735-9640
1572-9818
DOI:10.1007/BF02772830