![]() ![]() To illustrate this method we generated internal, N- and C-terminal deletions in the growth factor NT-3 previously cloned in the plasmid pcDNA6.2/GW/V5/D-TOPO. Performing a Dpn I digestion step ensures that all the transformants contain a mutated plasmid and not source DNA. After PCR, a linear DNA product is generated whose 5' and 3' ends contain the sequence of the restriction enzyme chosen and, after digestion and ligation, the DNA fragment is recircularized and used to transform cells. This restriction enzyme cutting sequence is introduced as non-complementary, non-overlapping strands in the 5' end of each primer. The deleted DNA sequence is replaced by the cutting sequence of a restriction enzyme plus the additional bases needed to keep the reading frame necessary to translate the DNA to protein. We describe in this manuscript a rapid PCR-based technique that takes advantage of the circularity of plasmid DNA to amplify the entire plasmid except for the region that is to be deleted. All those approaches require many different reactions, are not very efficient or reliable, and require repetition of the procedure many times before it successes. The most common approach to delete DNA sequences in plasmids uses naturally occurring restriction sites followed by ligation with the plasmid ( Allemandou et al. ![]() 2005), although the look for clones with the desired mutations could be time consuming and not very efficient. 1985) turned mutagenesis much simpler as many ingenious methods were developed, most of them based on introduction of mismatches in the primers to generate mutations ( Ling and Robinson 1995 Lanio and Jeltsch, 1998 Shenoy and Visweswariah, 2003 Chiu et al. The description of polymerase chain reaction (PCR) ( Saiki et al. The first mutagenesis methods had very low rate of success and were not cost effective ( Kramer et al. Site-directed mutagenesis is a powerful method commonly used in many areas of molecular biology and biochemistry to study the effect of point mutations in protein activity or to study the role of specific domains in protein function. Transfections and analysis of expression.Dpn I digestion, EcoRI digestion and ligation.The extraordinary simplicity of the method makes it a valuable tool to generate DNA deletions in plasmids and to study the effects of those deletions in protein function. The method is straightforward in its execution and success relies on a meticulous primer design that permits us obtain 100% of transformants containing the desired mutation. The only limitation is the selection of the restriction enzyme target sequence that must not be present in the original plasmid. Following PCR amplification, the plasmid is digested with Dpn I to eliminate the template DNA, with the chosen restriction enzyme, and ligated. The primers are designed to contain a non-complementary 5' sequence consisting of a restriction enzyme target sequence. We developed a simple, rapid and reliable method to delete DNA fragments in plasmids using a polymerase chain reaction based amplification of the circular DNA sequence that excludes the fragment to be deleted. ![]()
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