A Nested-Splicing by Overlap Extension PCR Improves Specificity of this Standard Method.

ABSTRACT:

Background

Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function.

Objectives

We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR.

Materials and methods

Genomic DNA from Bacillus thermocatenulatus was extracted. Nested PCR was used to amplify B. thermocatenulatus lipase gene variants, namely wild type and mutant, using gene specific and mutagenic specific primers, followed by cloning in a suitable vector. Briefly in N-SOE-PCR method, instead of two pairs of primers, three pairs of primers are used to amplify a mutagenic fragment. Moreover, the first a

SUBMITTER: Karkhane AA 

PROVIDER: S-EPMC5435007 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature