Directional genome walking using PCR.
Biotechniques
; 33(4): 830-2, 834, 2002 Oct.
Article
en En
| MEDLINE
| ID: mdl-12398192
We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5'-flanking genomic regions of many cDNA clones previously isolated by us.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Reacción en Cadena de la Polimerasa
/
Paseo de Cromosoma
/
Genoma de Planta
/
ADN de Plantas
Idioma:
En
Revista:
Biotechniques
Año:
2002
Tipo del documento:
Article
País de afiliación:
India
Pais de publicación:
Reino Unido