title: Use of Redundant Exclusion PCR To Identify a Novel Bacillus thuringiensis Cry8 Toxin Gene from Pooled Genomic DNA.
abstract: With 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 (RE-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 to be 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 Anomala corpulenta
  
  
  
  Protein toxins from the bacterium Bacillus thuringiensis are being increasingly used as biopesticides against a wide range of insect pests, yet the search for new or improved toxins is becoming more difficult, as traditional methods for gene discovery routinely isolate previously identified clones. This paper describes an approach that we have developed to increase the success rate for novel toxin gene identification through reducing or eliminating the cloning of previously characterized genes.
authors.affiliation: College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, People's Republic of China.|State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Fengjiao
authors.initials: F
authors.lastname: Zhang
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Changlong
authors.initials: C
authors.lastname: Shu
authors.affiliation: School of Life Sciences, University of Sussex, Brighton, United Kingdom.
authors.forename: Neil
authors.initials: N
authors.lastname: Crickmore
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Yanqiu
authors.initials: Y
authors.lastname: Li
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China.
authors.forename: Fuping
authors.initials: F
authors.lastname: Song
authors.affiliation: Cangzhou Academy of Agricultural and Forestry Sciences, Cangzhou, People's Republic of China.
authors.forename: Chunqin
authors.initials: C
authors.lastname: Liu
authors.affiliation: College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, People's Republic of China chenzhibao_2000@sina.com jzhang@ippcaas.cn.
authors.forename: Zhibao
authors.initials: Z
authors.lastname: Chen
authors.affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, People's Republic of China chenzhibao_2000@sina.com jzhang@ippcaas.cn.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000818:Animals; D001413:Bacillus thuringiensis / Q000235:genetics*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000633:toxicity; D001681:Biological Assay; D003001:Cloning, Molecular; D001517:Coleoptera / Q000187:drug effects; D017931:DNA Primers / Q000235:genetics; D004731:Endotoxins / Q000235:genetics* / Q000302:isolation & purification* / Q000633:toxicity; D015870:Gene Expression; D056726:Genetic Association Studies / Q000379:methods*; D006460:Hemolysin Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000633:toxicity; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D017931:DNA Primers; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis