Appl. Environ. Microbiol.-2016-Zhang-AEM.00862-16.pdf (2.74 MB)
Use of redundant exclusion PCR to identify a novel Bacillus thuringiensis Cry8 toxin gene from pooled genomic DNA
journal contribution
posted on 2023-06-09, 00:57 authored by Fengjiao Zhang, Changlong Shu, Neil CrickmoreNeil Crickmore, Yanqiu Li, Fuping Song, Chunqin Liu, Zhibao Chen, Jie ZhangWith the aim of optimizing the cloning of novel genes from a genomic pool containing many previously identified, homologous, genes we designed a redundant exclusion PCR technique. In RE-PCR a pair of generic amplification primers are combined with additional primers that are designed to specifically bind to redundant, unwanted genes that are a subset of those copied by the amplification primers. During RE-PCR the specific primer blocks amplification of the full length redundant gene. Using this method we managed to clone a number of cry8 or cry9 toxin genes from a pool of Bacillus thuringiensis genomic DNA while excluding amplicons for cry9Da, cry9Ea and cry9Eb. The method proved very efficient at increasing the number of rare genes in the resulting library. One such rare, and novel, cry8-like gene was expressed and the encoded toxin was shown to be toxic to Anomola corpulenta.
History
Publication status
- Published
File Version
- Accepted version
Journal
Applied and Environmental MicrobiologyISSN
0099-2240Publisher
American Society for MicrobiologyExternal DOI
Issue
13Volume
82Page range
3808-3815Department affiliated with
- Biochemistry Publications
Full text available
- Yes
Peer reviewed?
- Yes
Legacy Posted Date
2016-04-19First Open Access (FOA) Date
2016-04-19First Compliant Deposit (FCD) Date
2016-04-19Usage metrics
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