Journal
AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY
Volume 288, Issue 6, Pages C1273-C1278Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajpcell.00517.2004
Keywords
type IIs restriction enzyme; enhanced green fluorescent protein; Bcl-2
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Funding
- NCI NIH HHS [R01 CA-95739] Funding Source: Medline
- NHLBI NIH HHS [R01 HL-69000] Funding Source: Medline
- NIA NIH HHS [R01 AG-15556] Funding Source: Medline
- NIDDK NIH HHS [R01 DK-51770] Funding Source: Medline
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PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.
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