Utilize este identificador para referenciar este registo:
https://hdl.handle.net/1822/45774
Título: | Inverse PCR for point mutation introduction |
Autor(es): | Silva, Diogo Santos, Gustavo Barroca, Mário Jorge Faria Collins, Tony |
Editor(es): | Domingues, Lucília |
Palavras-chave: | Inverse PCR Nonoverlapping primers Protein engineering Site-directed mutagenesis |
Data: | 25-Mai-2017 |
Editora: | Springer Verlag |
Revista: | Methods in Molecular Biology |
Resumo(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. |
Tipo: | Capítulo de livro |
URI: | https://hdl.handle.net/1822/45774 |
ISBN: | 978-1-4939-7059-9 |
e-ISBN: | 978-1-4939-7060-5 |
DOI: | 10.1007/978-1-4939-7060-5_5 |
ISSN: | 1064-3745 |
Versão da editora: | https://link.springer.com/protocol/10.1007%2F978-1-4939-7060-5_5 |
Acesso: | Acesso restrito autor |
Aparece nas coleções: | DBio - Livros e Capítulos de Livros/Books and Books Chapters |
Ficheiros deste registo:
Ficheiro | Descrição | Tamanho | Formato | |
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Silva et al iPCR for SDM Corrected 20170223.pdf Acesso restrito! | 204,88 kB | Adobe PDF | Ver/Abrir |