Abstract：
:A simple, two-step efficient method to perform multiple-site mutagenesis of a gene from bacterial genome was developed. The method was named polyacrylamide gel electrophoresis (PAGE)-mediated overlap extension polymerase chain reaction (PCR) (POEP). The first step involves synthesis of individual fragments containing mutant sites with 15- to 25-bp overlap between two adjacent fragments. Mutations were introduced into the overlapping oligonucleotide primers which ensured the particular primer-template annealing. PAGE was used to remove contaminating parental templates, mispriming fragments, and leftover primers. The second step involves synthesis of the mutant full-length fragment. All purified PCR products from the first step were combined and used as the template for a second PCR using high-fidelity DNA polymerase, with the two outermost flanking oligonucleotides as primers. Using the POEP method, we have successfully introduced eight EcoRI sites into the Escherichia coli beta-galactosidase (Lac Z) gene. The overall rate of obtaining the multiple mutant sites was 100%. The POEP method is simple, involving only two steps, and reliable for multiple-site mutagenesis and is promising to be widely used in gene modification.
journal_name
Appl Microbiol Biotechnoljournal_title
Applied microbiology and biotechnologyauthors
Peng RH,Xiong AS,Yao QHdoi
10.1007/s00253-006-0583-3subject
Has Abstractpub_date
2006-11-01 00:00:00pages
234-40issue
1eissn
0175-7598issn
1432-0614journal_volume
73pub_type
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