Please use this identifier to cite or link to this item: http://hdl.handle.net/1822/45774

TitleInverse PCR for point mutation introduction
Author(s)Silva, Diogo
Santos, Gustavo
Barroca, Mário Jorge Faria
Collins, Tony
Editor(s)Domingues, Lucília
KeywordsInverse PCR
Nonoverlapping primers
Protein engineering
Site-directed mutagenesis
Issue date25-May-2017
PublisherSpringer Verlag
JournalMethods in Molecular Biology
Abstract(s)Inverse PCR is a powerful tool for the rapid introduction of desired mutations at desired positions in a circular double-stranded DNA sequence. Here, custom-designed mutant primers oriented in the inverse direction are used to amplify the entire circular template with incorporation of the required mutation(s). By careful primer design it can be used to perform such diverse modifications as the introduction of point mutations and multiple mutations, the insertion of new sequences, and even sequence deletions. Three primer formats are commonly used; nonoverlapping, partially overlapping and fully overlapping primers, and here we describe the use of nonoverlapping primers for introduction of a point mutation. Use of such a primer setup in the PCR reaction, with one of the primers containing the desired mismatch mutation, results in the amplification of a linear, double-stranded, mutated product. Methylated template DNA is removed from the nonmethylated PCR product by DpnI digestion and the PCR product is then phosphorylated by polynucleotide kinase treatment before being recircularized by ligation, and transformed to E. coli. This relatively simple site-directed mutagenesis procedure is of major importance in biology and biotechnology today where it is commonly employed for the study and engineering of DNA, RNA, and proteins.
TypeBook part
URIhttp://hdl.handle.net/1822/45774
ISBN978-1-4939-7059-9
e-ISBN978-1-4939-7060-5
DOI10.1007/978-1-4939-7060-5_5
ISSN1064-3745
Publisher versionhttps://link.springer.com/protocol/10.1007%2F978-1-4939-7060-5_5
AccessRestricted access (Author)
Appears in Collections:DBio - Livros e Capítulos de Livros/Books and Books Chapters

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