Display and isolation of transposon-flanking sequences starting from genomic DNA or RNA

Autor: Alexander Yephremov, Heinz Saedler
Rok vydání: 2000
Předmět:
Zdroj: The Plant Journal. 21:495-505
ISSN: 1365-313X
0960-7412
DOI: 10.1046/j.1365-313x.2000.00704.x
Popis: Insertion mutagenesis using transposons has become a powerful tool for the isolation of genes involved in any given biochemical or developmental pathway. We describe here ligation-mediated PCR techniques for the isolation of sequences flanking the transposable elements En/Spm, Mu1 and Cin4 in Zea mays and Arabidopsis thaliana. Two versions of this transposon insertion display (TID) method use biotinylated linkers or biotinylated primers to rapidly isolate transposon-flanking sequences starting from digested genomic DNA. TID protocols have been employed to clone several genes from En/Spm insertion mutants of Arabidopsis. A novel procedure, expression TID (ETID), is also introduced, which provides a direct approach for the isolation of transposon insertions that tag transcribed portions of genes. ETID uses RNA as a starting material and exploits 5' RACE PCR to identify transposon copies that form parts of gene transcripts. The detection of several En/Spm insertion mutations in Arabidopsis illustrates the power of this method. ETID offers important advantages for the isolation of mutant alleles of novel genes that are expressed in specific tissues in plants and animals.
Databáze: OpenAIRE
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