Patent ID: 7452700
Filing Date: 2008-11-18
Classification: C12Q

Abstract:
1. A method for identifying novel homologues of a target group of pesticidal genes of interest, said method comprising: a) designing at least one pair of oligonucleotide primers that is specific for said target group of pesticidal genes, said pair of primers comprising a forward primer and a reverse primer, wherein said designing comprises wherein, EH o (enthalpy)=ΣΔH; ES o (entropy)=ΣΔS+0.368×19×1.585; R (molar gas constant)=1.987; Ct (total primer concentration)=log(0.00000005/4)×1000; and, X (salt concentration [K+])=0.05; wherein a nucleotide sequence that is not conserved among non-target group pesticidal genes differs from each of the non-target group pesticidal genes by at least two nucleotide residues; b) obtaining a first sample of nucleic acid material from a microorganism of interest; c) mixing said first sample of nucleic acid material with said at least one pair of oligonucleotide primers specific for said target group of pesticidal genes and a thermostable DNA polymerase under conditions that are suitable for amplification by polymerase chain reaction (PCR); d) performing a first round of PCR and detecting PCR amplification products, thereby determining if PCR products are produced in the first round of PCR; e) obtaining a second sample of nucleic acid material from the microorganism if PCR amplification products are detected in the first round of PCR; f) subjecting the second sample of nucleic acid material to a second round of amplification by PCR using pairs of oligonucleotide primers that are specific for all known pesticidal genes in the target group, wherein said pairs of oligonucleotide primers specific for known pesticidal genes in the target group comprise nucleotide sequences that are different from the nucleotide sequences for said oligonucleotide primers of (a); g) detecting PCR amplification products from the second round of PCR, thereby determining if PCR products are produced in the second round of PCR; h) obtaining a third sample of nucleic acid material from the microorganism if PCR products are detected in the first round of PCR and PCR products are not detected in the second round of PCR, wherein a microorganism that comprises nucleic acid material that is amplified in the first round of PCR and is not amplified in the second round of PCR comprises a putative novel homologue of the target group of pesticidal genes; i) subjecting the third sample of nucleic acid material to a third round of PCR using at least one pair of oligonucleotide primers to clone the putative novel homologue; and, j) analyzing the putative novel homologue of the target group of pesticidal genes of interest.