Reverse genetics using insertional mutagenesis

o Create insertion library using transposons (any organism) or T-DNA (plants) 2 3 1 100,000 9 Isolate DNA _ . \ - Transposable 9 PCR with primers g1, 92, t1, t2 Example of Insertion element/T—DNA Wild type allele Inserted Into (no insertion in gene) Insertion mutant exon 2 of a gene. — — ‘ g1+gZ gl+t1 gZ+t2 g1+ngl+t1gZ+t2 fi’ Gene—specific primers (91 and 92) are used in conjuction with tranposorVT—DNA—specific primers (t1 and t2) in PCR reactions. If a gene does not If a gene has an insertion, specific have insertion (wild combinations of g and t primers type), only the (in this case 91 + T1 and 92 + t2) combination of will yield a product. In addition, primers gi + 92 the 91 + 92 primers should yield results in a product. a larger product as compared to wild type.