An improved PCR-based method for site directed mutagenesis using megaprimers

TY - JOUR

T1 - An improved PCR-based method for site directed mutagenesis using megaprimers

AU - Brøns-Poulsen, J

AU - Petersen, N E

AU - Hørder, M

AU - Kristiansen, K

N1 - Keywords: DNA Primers; DNA-Directed DNA Polymerase; Humans; Hydroxymethylbilane Synthase; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction

PY - 1998

Y1 - 1998

N2 - An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.

AB - An improved protocol for site-directed mutagenesis based on the two-step polymerase chain reaction (PCR) megaprimer method is described. Compared to previously published protocols, the protocol described in this article ensures consistently a success rate of at least 85% with essentially no introduction of unwanted secondary mutations. The essential features of this protocol include an optimization of the template-primer amounts and ratio that allows the use of a reduced number of PCR cycles and the use of proof-reading thermostable DNA polymerases.

U2 - 10.1006/mcpr.1998.0187

DO - 10.1006/mcpr.1998.0187

M3 - Journal article

C2 - 9843651

SN - 0890-8508

VL - 12

SP - 345

EP - 348

JO - Molecular and Cellular Probes

JF - Molecular and Cellular Probes

IS - 6

ER -