Method for identifying novel genes

A method for identifying novel genes that share homology with a target group of known pesticidal genes, the method comprising:

• a) designing at least one pair of oligonucleotide primers for use in a first round of PCR that is specific for the target group of pesticidal genes, the pair of primers comprising a forward primer and a reverse primer, wherein each primer targets a signature sequence present in the nucleotide sequences of the target group, wherein said designing comprises
• i) preparing an alignment of all nucleotide sequences from the target group;
• ii) identifying signature sequences within the nucleotide sequences of the target group of pesticidal genes, wherein a signature sequence comprises a region of homology between all members of the target group;
• iii) selecting an initial primer length, wherein the initial primer length is between 15 bp and 30 bp;
• iv) performing a first round of screening for an oligonucleotide primer sequence, the screening comprising viewing an initial window of contiguous nucleotides of a signature sequence within the nucleotide sequences of the target group, wherein the initial window is initiated at the 5′ end of the nucleotide sequence of the signature sequence;
• v) determining if the nucleotide sequence within the initial window has the sequence features of (1)-(6) below
• (1) does not have four or more contiguous identical nucleotide residues;
• (2) has no more than two guanine or cytosine residues within the last five residues of the 3′ end of the nucleotide sequence;
• (3) has a Tm between 50° C. and 65° C.;
• (4) does not form hairpin or dimer structures;
• (5) is present in at least one of the nucleotide sequences from the target group of pesticidal genes;
• and
• (6) is not conserved among nucleotide sequences from non-target group pesticidal genes;
• wherein one nucleotide residue within the initial window of contiguous nucleotide sequences is permitted to be n, wherein n is any nucleotide selected from the group consisting of adenine, thymine, guanine, and cytosine;
• vi) selecting the nucleotide sequence within the initial window for use as an oligonucleotide primer if all of the sequence features of step (v) are present;
• vii) selecting an adjacent window of contiguous nucleotides by moving the first window toward the 3′ end of the signature sequence within the nucleotide sequences of the target group by one base pair if the nucleotide sequence within the initial window does not have all of the sequence features of step v), wherein the adjacent window is equivalent in length to the initial primer length;
• viii) repeating steps v)-vii) with the adjacent window until a nucleotide sequence with all of the sequence features is identified or until the entire signature sequence for the target group is screened;
• and
• ix) selecting a second signature sequence within the nucleotide sequences of the target group of pesticidal genes and performing additional rounds of screening comprising repeating steps iii) through viii) using the second signature sequence if no nucleotide sequence with all of the features is identified by screening the first signature sequence;
• b) obtaining a first sample of nucleic acid material from an organism of interest;
• c) mixing the first sample of nucleic acid material with the at least one pair of oligonucleotide primers for use in the first round of PCR and a thermostable DNA polymerase under conditions that are suitable for amplification by 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 organism if PCR products are detected in the first round of PCR;
• f) designing at least one pair of oligonucleotide primers for use in a second round of PCR that is specific for the target group of pesticidal genes, the pair of primers comprising a forward primer and a reverse primer, wherein each primer targets a signature sequence present in the nucleotide sequences of the target group, and wherein said at least one pair of oligonucleotide primers for use in the second round of PCR does not comprise the nucleotide sequences of said at least one pair of oligonucleotide primers of (a);
• g) mixing the second sample of nucleic acid material with the at least one pair of oligonucleotide primers for use in the second round of PCR and a thermostable DNA polymerase under conditions that are suitable for amplification by PCR and performing a second round of PCR;
• h) separating any PCR amplification products produced in the second round of PCR using agarose gel electrophoresis and isolating nucleic acid fragments for further analysis, wherein the nucleic acid fragments may comprise a putative novel gene fragment that shares homology with the genes of the target group of pesticidal genes;
• i) cloning each nucleic acid fragment into a cloning vector;
• j) transforming host cells with the cloning vectors, wherein the cloning vectors comprise the nucleic acid fragments isolated in step (h);
• k) preparing nucleic acid samples from individual host colonies comprising a cloning vector;
• l) subjecting the nucleic acid samples from the individual host colonies to dot blot analysis using labeled probes that are specific for all known genes from the target group, wherein a nucleic acid fragment isolated in step (h) that is not detected during the dot blot analysis step comprises a putative novel gene fragment that shares homology with the genes of the target group;
• and
• m) analyzing the putative novel gene fragment.

A method for identifying novel pesticidal genes, the method comprising:

• a) designing at least one pair of oligonucleotide primers for use in a first round of PCR that is specific for a target group of pesticidal genes, the pair of primers comprising a forward primer and a reverse primer, wherein each primer targets a signature sequence present in the nucleotide sequences of the target group;
• b) obtaining a first sample of nucleic acid material from a microorganism of interest;
• c) mixing the first sample of nucleic acid material with the at least one pair of oligonucleotide primers for use in the first round of PCR and a thermostable DNA polymerase under conditions that are suitable for amplification by 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, wherein the first round of PCR comprises performing quantitative real-time PCR;
• e) obtaining a second sample of nucleic acid material from the microorganism if PCR products are detected in the first round of PCR;
• f) designing at least one pair of oligonucleotide primers for use in a second round of PCR that is specific for the target group of pesticidal genes, the pair of primers comprising a forward primer and a reverse primer, wherein each primer targets a signature sequence present in the nucleotide sequences of the target group, and wherein said at least one pair of oligonucleotide primers for use in the second round of PCR does not comprise the nucleotide sequences of said at least one pair of oligonucleotide primers of (a);
• g) mixing the second sample of nucleic acid material with the at least one pair of oligonucleotide primers for use in the second round of PCR and a thermostable DNA polymerase under conditions that are suitable for amplification by PCR and performing a second round of PCR;
• h) separating any PCR amplification products produced in the second round of PCR using agarose gel electrophoresis and isolating nucleic acid fragments for further analysis, wherein the nucleic acid fragments may comprise a putative novel gene fragment that shares homology with the pesticidal genes of the target group;
• i) cloning each nucleic acid fragment into a cloning vector;
• j) transforming host cells with the cloning vectors, wherein the cloning vectors comprise the nucleic acid fragments isolated in step (h);
• k) preparing nucleic acid samples from individual host colonies comprising a cloning vector;
• l) subjecting the nucleic acid samples from the individual host colonies to dot blot analysis using labeled probes that are specific for all known pesticidal genes from the target group, wherein a nucleic acid fragment isolated in step (h) that is not detected during the dot blot analysis step comprises a putative novel pesticidal gene fragment;
• and
• m) analyzing the putative novel pesticidal gene fragment.

A method for identifying novel pesticidal genes, the method comprising:

• a) designing at least one pair of oligonucleotide primers for use in a first round of PCR that is specific for a target group of pesticidal genes, the pair of primers comprising a forward primer and a reverse primer, wherein each primer targets a signature sequence present in the nucleotide sequences of the target group;
• b) obtaining a first sample of nucleic acid material from a microorganism of interest;
• c) mixing the first sample of nucleic acid material with the at least one pair of oligonucleotide primers for use in the first round of PCR and a thermostable DNA polymerase under conditions that are suitable for amplification by 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 products are detected in the first round of PCR;
• f) designing at least one pair of oligonucleotide primers for use in a second round of PCR that is specific for the target group of pesticidal genes, the pair of primers comprising a forward primer and a reverse primer, wherein each primer targets a signature sequence present in the nucleotide sequences of the target group, and wherein said at least one pair of oligonucleotide primers for use in the second round of PCR does not comprise the nucleotide sequences of said at least one pair of oligonucleotide primers of (a);
• g) mixing the second sample of nucleic acid material with the at least one pair of oligonucleotide primers for use in the second round of PCR and a thermostable DNA polymerase under conditions that are suitable for amplification by PCR and performing a second round of PCR;
• h) separating any PCR amplification products produced in the second round of PCR using agarose gel electrophoresis and isolating nucleic acid fragments for further analysis, wherein the nucleic acid fragments may comprise a putative novel pesticidal gene fragment;
• i) cloning each nucleic acid fragment into a cloning vector;
• j) transforming host cells with the cloning vectors, wherein the cloning vectors comprise the nucleic acid fragments isolated in step (h);
• k) preparing nucleic acid samples from individual host colonies comprising a cloning vector;
• l) subjecting the nucleic acid samples from the individual host colonies to dot blot analysis using labeled probes that are specific for all known pesticidal genes from the target group, wherein a nucleic acid fragment isolated in step (h) that is not detected during the dot blot analysis step comprises a putative novel pesticidal gene fragment;
• and
• m) analyzing the putative novel pesticidal gene fragment;
• wherein the Tm for the at least one pair of oligonucleotide primers used in the first and second rounds of PCR is 50° C. to 65° C.