BioMed Central Page 1 of 8 (page number not for citation purposes) BMC Biotechnology Open Access Methodology article Simple and efficient site-directed mutagenesis using two single-primer reactions in parallel to generate mutants for protein structure-function studies Oded Edelheit 1,2,3 , Aaron Hanukoglu 2,3 and Israel Hanukoglu* 1 Address: 1 Department of Molecular Biology, Ariel University Center, Ariel, Israel, 2 Department of Pediatrics, Tel-Aviv University, Sackler Medical School, Tel Aviv, Israel and 3 Division of Pediatric Endocrinology, E. Wolfson Medical Center, Holon, Israel Email: Oded Edelheit - ede.oded@gmail.com; Aaron Hanukoglu - aaronh@science.co.il; Israel Hanukoglu* - mbiochem@gmail.com * Corresponding author Abstract Background: In protein engineering, site-directed mutagenesis methods are used to generate DNA sequences with mutated codons, insertions or deletions. In a widely used method, mutations are generated by PCR using a pair of oligonucleotide primers designed with mismatching nucleotides at the center of the primers. In this method, primer-primer annealing may prevent cloning of mutant cDNAs. To circumvent this problem we developed an alternative procedure that does not use forward-reverse primer pair in the same reaction. Results: In initial studies we used a double-primer PCR mutagenesis protocol, but sequencing of products showed tandem repeats of primer in cloned DNA. We developed an alternative method that starts with two Single-Primer Reactions IN Parallel using high-fidelity Pwo DNA polymerase. Thus, we call the method with the acronym SPRINP. The SPRINP reactions are then combined, denatured at 95°C, and slowly cooled, promoting random annealing of the parental DNA and the newly synthesized strands. The products are digested with DpnI that digests methylated parental strands, and then transformed into E. coli. Using this method we generated >40 mutants in cDNAs coding for human Epithelial Na + Channel (ENaC) subunits. The method has been tested for 1–3 bp codon mutation and insertion of a 27 bp epitope tag into cDNAs. Conclusion: The SPRINP mutagenesis protocol yields mutants reliably and with high fidelity. The use of a single primer in each amplification reaction increases the probability of success of primers relative to previous methods employing a forward and reverse primer pair in the same reaction. Background Site-directed mutagenesis (SDM) methods are used to generate cloned DNAs with modified sequences for exam- ining the importance of specific residues in protein struc- ture and function. SDM represents the primary rational method in protein engineering and for altering enzyme substrate selectivity [1,2]. Currently prevalent methods of SDM employ PCR using oligonucleotide primer pairs that carry the desired muta- tion. There is a large variety of approaches for SDM using PCR [1,3,4]. One of the widely used methods is Quik- Change [5]. This method and its later modifications employ complementary primer pairs in the same PCR reaction. The use of complementary primer pairs may lead Published: 30 June 2009 BMC Biotechnology 2009, 9:61 doi:10.1186/1472-6750-9-61 Received: 26 February 2009 Accepted: 30 June 2009 This article is available from: http://www.biomedcentral.com/1472-6750/9/61 © 2009 Edelheit et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.