“megaprimer method” of site-directed mutagenesis. BioTechniques 8:404-407. 11.Senanayake, S.D. and D.A. Brian. 1995. Precise large deletions by the PCR based overlap extention. Mol. Biotechnol. 4:13-15. 12.Silver, J., T. Limjoco, and Feinstone. 1995. Site-specific mutagenesis using the polymerase chain reaction, p. 179-188. In M.A. Innis, D.H. Gelfand and J.J. Sninsky (Eds.), PCR Strategies. Ac...

tenecteplase is a variant of tissue plasminogen activator (t-pa) which has better pharmacokinetic properties and more selective thrombolytic activity. in the present study, we describe a rapid method to introduce three sets of mutation into defined positions in t-pa cdna by a site-directed mutagenesis based on a megaprimer pcr approach to produce tenecteplase coding sequence where amino acids a...

The "megaprimer" method (1) based on polymerase chain reaction (PCR) is one of the simplest and most versatile procedures of site-specific in vitro mutagenesis available to date. The method utilizes three oligonucleotide primers and two rounds of PCR performed on a DNA template containing the cloned gene that is to be mutated. The rationale of the method is shown schematically in Fig. 1 where A...

A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total num...

It is well known that some genetic mutations have a critical impact on protein structure and function. To study the effects of these mutations, site-directed mutagenesis allows the introduction of a defined substitution of specific residues. For the past few years, numerous PCR-based methods have been employed for achieving site-directed mutagenesis. Among these methods, megaprimer PCR has been...

Site-directed mutagenesis has become an essential tool in the investigation of gene structure and function as well as the products of gene expression, namely RNA and protein. Selected positions in a DNA sequence are changed, and the eVects of the mutations are studied in vitro and/or in vivo. Although many diVerent mutagenic strategies are available [1], the simplicity, cost eVectiveness, and a...

Efficient site-directed mutagenesis (SDM) is a powerful tool for the analysis of the function of DNA sequences. The polymerase chain reaction (PCR) (4,9) and commercially available methods for in vitro selection of mutated unmethylated DNA (Stratagene, La Jolla, CA, USA) have been used increasingly as rapid alternatives to classical methods of site-specific mutation (11). Generating mutations a...

The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to...