Fusion PCR and gene targeting in Aspergillus nidulans.
Nat Protoc
; 1(6): 3111-20, 2006.
Article
en En
| MEDLINE
| ID: mdl-17406574
ABSTRACT
We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.
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Bases de datos:
MEDLINE
Asunto principal:
Aspergillus nidulans
/
Reacción en Cadena de la Polimerasa
/
Marcación de Gen
Idioma:
En
Revista:
Nat Protoc
Año:
2006
Tipo del documento:
Article
País de afiliación:
Estados Unidos