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WO2002040677A2 - Evenement du coton pv-ghbk04 (531) et compositions et procedes permettant de detecter la presence de ce dernier - Google Patents

Evenement du coton pv-ghbk04 (531) et compositions et procedes permettant de detecter la presence de ce dernier Download PDF

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Publication number
WO2002040677A2
WO2002040677A2 PCT/US2001/043297 US0143297W WO0240677A2 WO 2002040677 A2 WO2002040677 A2 WO 2002040677A2 US 0143297 W US0143297 W US 0143297W WO 0240677 A2 WO0240677 A2 WO 0240677A2
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Prior art keywords
seq
dna
sequence
event
cotton
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PCT/US2001/043297
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English (en)
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WO2002040677A3 (fr
Inventor
Kim A. Beazley
Jeanna R. Hillyard
Shengzhi Pang
James K. Roberts
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Monsanto Technology Llc
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Application filed by Monsanto Technology Llc filed Critical Monsanto Technology Llc
Priority to US10/416,877 priority Critical patent/US20040045054A1/en
Priority to AU2002236442A priority patent/AU2002236442A1/en
Publication of WO2002040677A2 publication Critical patent/WO2002040677A2/fr
Publication of WO2002040677A3 publication Critical patent/WO2002040677A3/fr
Priority to US11/801,114 priority patent/US7964348B2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

Definitions

  • the present invention relates to the field of plant molecular biology, more specifically the invention relates to cotton event 531, and to assays for detecting the presence of cotton event 531 in a sample.
  • This invention relates to the lepidopteran resistant cotton (Gossypium hirsutum) plant 531 and to the detection of the transgene/genomic insertion regions in cotton plant 531 and progeny thereof.
  • the present invention relates to the field of plant molecular biology, more specifically the invention relates to identification of nucleic acids from the transgenic cotton event 531, preferably to assays for detecting the presence of cotton event 531 in a sample and compositions thereof.
  • Cotton is an important fiber crop in many areas of the world. The methods of biotechnology have been applied to cotton for improvement of the agronomic traits and the quality of the product. The method of introducing transgenes into cotton plants is demonstrated in U.S. Patent No. 5,004,863.
  • nucleic acid detection method such as nucleic acid amplification techniques or nucleic acid hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc.
  • compositions and methods are provided for detecting the presence of various transgene/genomic insertion regions from a cotton plant designated PN-GHBK04, also known herein as cotton event 531.
  • D ⁇ A sequences are provided that comprise at least one junction sequence of 531 identified as SEQ ID ⁇ O:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:32, and complements thereof; wherein a junction sequence spans the junction between heterologous DNA inserted into the genome and the DNA from the cotton cell flanking the insertion site and is diagnostic for the event.
  • This invention relates to the seeds and to the progeny of cotton event 531, and to methods for detecting nucleic acids contained within and produced by the event 531 in a biological or commercial sample.
  • methods of producing a lepidopteran resistant cotton plant comprise the steps of: (a) sexually crossing a first parental cotton line comprising cotton event 531 DNA that exhibits a trait which confers resistance to one or more lepidopteran insect species upon the event, with a second parental cotton line that does not exhibit lepidopteran insect resistance, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that exhibits resistance to one or more lepidopteran insect species.
  • the methods are useful for introgressing the lepidopteran resistance trait into different genetic backgrounds. Such methods may optionally comprise a further step of back-crossing the progeny plant to the second parental cotton line to produce a cotton plant that is also lepidopteran resistant.
  • the DNA sequences that comprise at least 11 or more contiguous nucleotides of the DNA sequence of SEQ ID NO: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 and complements thereof for use as DNA primers in DNA amplification methods.
  • the amplicons produced using these primers are diagnostic for cotton event 531.
  • the amplicons produced by said DNA primers are an aspect of the invention.
  • methods of detecting the presence of DNA corresponding to the cotton event 531 event in a sample comprise: (a) contacting the sample comprising DNA with a primer set that, when used in a nucleic acid amplification reaction with DNA from cotton event 531, produces an amplicon that is diagnostic for cotton event 531 nucleic acids in a sample; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon.
  • methods of detecting the presence of a primer set that, when used in a nucleic acid amplification reaction with DNA from cotton event 531, produces an amplicon that is diagnostic for cotton event 531 nucleic acids in a sample.
  • methods of detecting the presence of a primer set that, when used in a nucleic acid amplification reaction with DNA from cotton event 531, produces an amplicon that is diagnostic for cotton event 531 nucleic acids in a sample
  • DNA corresponding to the 531 event in a sample comprising: (a) contacting the sample comprising DNA with a probe that hybridizes under stringent hybridization conditions with DNA from cotton event 531 (i.e., does not hybridize to nucleic acid sequences which are other than DNA from cotton event 531) and does not hybridize under the stringent hybridization conditions with a control cotton plant (non-531 DNA); (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the DNA.
  • Figure 1 is a schematic representation of the inserted heterologous DNA in cotton event 531 representing the arrangement of the DNA inserted at a single genome locus and containing (i) A, at the indicated 3' end a cassette comprising a full length functional inserted DNA sequence from plasmid PV-GHBK04 including an nptll coding sequence and a cry 1 A coding sequence, and at the indicated 5' end a partial crylA coding sequence and 7S 3' termination sequence inverted with reference to the sequence within the full length functional inserted DNA sequence; and (ii) B, a partial 7S 3' sequence which is not physically linked to the cotton event 531; wherein vertical serrated lines indicate junctions between heterologous inserted DNA and plant genomic DNA, arrows within the genetic elements imply the direction of transcription orientation, and triangles represent the border region of T-DNA sequences.
  • Figure 2 illustrates the results of thermal amplification analysis of the 5' and 3' insert to plant junctions of the full length functional inserted DNA in cotton event 531, performed on genomic DNA isolated from cotton event 531 seed tissue by pairing an insert and a flanking sequence primer specific to the 5 '-end of the insert (Panel A, Primers C and D, respectively) or the 3 '-end of the insert (Panel B, Primers A and B, respectively); Lanes 2, 3 and 4 contain the product generated from cotton event 531 genomic DNA template, control reactions containing no template DNA (Lane 6), and Coker 312 non-transgenic control DNA (Lane 5); the location of the primers used in the analyses appear as small arrows below the illustration of the inserted DNA below the panels; triangles represent the T-DNA sequence.
  • Figure 3 illustrates the results of thermal amplification analysis of a portion of the 7S 3' sequence in cotton event 531, performed on genomic DNA isolated from cotton event 531 seed tissue by pairing an insert and a flanking sequence primer specific to the 5 '-end of the insert (Panel A, Primers E and F, respectively) or the 3 '-end of the insert (Panel B, Primers G and H, respectively); primers are denoted as small arrows below the diagram of the inserted DNA below the panels; Lanes 2, 3 and 4 contain the product generated from genomic DNA template of cotton event 531 ; control reactions containing no template (Lane 6), and Coker 312 non- transgenic control DNA (Lane 5); triangle represents T-DNA sequence.
  • SEQ ID NO: l corresponds to a junction sequence which is diagnostic for the arbitrarily assigned 3' end of the full length primary functional inserted DNA sequence in cotton event 531.
  • SEQ ID NO:2 corresponds to a junction sequence which is diagnostic for the arbitrarily assigned 5' end of the partial cry 1 Ac DNA coding sequence inserted in cotton event 531.
  • SEQ ID NO: 3 corresponds to a junction sequence which is diagnostic for the arbitrarily assigned 5' end of the full length primary functional inserted DNA sequence in cotton event 531.
  • SEQ ID NO:4 corresponds to a junction sequence which is diagnostic for the arbitrarily assigned 3' end of the partial 7S 3' plant transcription termination and polyadenylation sequence inserted into the genome in cotton event 531.
  • SEQ ID NO: 5 corresponds to the sequence of the arbitrarily assigned 5' end of the partial cry 1 Ac DNA coding sequence inserted in cotton event 531.
  • SEQ ID NO: 6 corresponds to the cotton genome DNA sequence which is adjacent to and flanking the 5' end of the arbitrarily assigned 5' end of the partial cry 1 Ac DNA coding sequence inserted in cotton event 531.
  • SEQ ID NO: 7 corresponds to cotton genome DNA sequence which is adjacent to and flanking the 5' end of the full length primary functional inserted DNA sequence in cotton event 531.
  • SEQ ID NO: 8 corresponds to the DNA sequence of the arbitrarily assigned 5' end of the full length primary functional inserted DNA in cotton event 531.
  • SEQ ID NO: 9 corresponds to the cotton genome sequence adjacent to and flanking the arbitrarily assigned 3' end of the partial 7S 3' plant transcription termination and polyadenylation sequence inserted into the genome in cotton event 531.
  • SEQ ID NO: 10 corresponds to DNA sequence of the arbitrarily assigned 3' end of the full length primary functional inserted DNA in cotton event 531.
  • SEQ ID NO: 11 corresponds to the cotton genome DNA sequence adjacent to and flanking the arbitrarily assigned 3' end of the full length primary functional inserted DNA in cotton event 531.
  • SEQ ID NO: 12 corresponds to a primer sequence complementary to a part of the cotton genomic DNA sequence identified as flanking the arbitrarily assigned 3' end of the full length primary functional inserted DNA sequence in cotton event 531, and produces amplicon diagnostic for cotton event 531 DNA in a sample when paired with a primer corresponding to the sequence set forth in SEQ ID NO: 13 and cotton event 531 template DNA.
  • SEQ ID NO: 13 corresponds to a primer sequence complementary to a part of the arbitrarily assigned 3' end sequence of the full length primary functional DNA inserted into the cotton genome in cotton event 531, and produces an amplicon diagnostic for cotton event 531
  • SEQ ID NO: 14 corresponds to a sequence diagnostic for cotton event 531 DNA in a biological sample.
  • SEQ ID NO: 15 corresponds to a sequence diagnostic for cotton event 531 DNA in a biological sample.
  • SEQ ID NO: 16 corresponds to a sequence diagnostic for cotton event 531 DNA in a biological sample. 1 -
  • SEQ ID NO: 17 corresponds to Primer A, a sequence which is or is complementary to a sequence within the arbitrarily assigned 3' end of the full length functional inserted DNA in event 531.
  • SEQ ID NO: 18 corresponds to Primer B, a sequence which is or is complementary to a sequence within the 3' end flanking cotton genome sequence near the arbitrarily assigned 3' end of the full length functional inserted DNA in event 531.
  • SEQ ID NO: 19 corresponds to Primer C, a sequence which is or is complementary to a sequence within the sequence in event 531 which is arbitrarily assigned as the 5' end of the inserted sequence linked to the full length functional inserted DNA.
  • SEQ ID NO:20 corresponds to Primer D, a sequence which is or is complementary to a sequence within a part of the 5' end flanking cotton genome sequence near the arbitrarily assigned 5' end of the partial crylA coding sequence, which is linked 5' to the full length functional inserted DNA in event 531.
  • SEQ ID NO:21 corresponds to Primer E, a sequence which is or is complementary to the arbitrarily assigned 3' end of the partial 7S 3' sequence inserted into the cotton genome in event 531.
  • SEQ ID NO. -22 corresponds to Primer F, a sequence which is or is complementary to a part of the 5' end flanking cotton genome sequence near the arbitrarily assigned 5' end of the partial 7S 3' sequence inserted into the cotton genome in event 531.
  • SEQ ID NO:23 corresponds to Primer G, a sequence which is or is complementary to the arbitrarily assigned 5' end of the partial 7S 3' sequence inserted into the cotton genome in event 531.
  • SEQ ID NO:24 corresponds to Primer H, a sequence which is or is complementary to a part of the 3' end flanking cotton genome sequence near the arbitrarily assigned 3' end of the partial 7S 3' sequence inserted into the cotton genome in event 531.
  • SEQ ID NO:25 corresponds to Primer I, a sequence which is or is complementary to a part of the 5' end flanking cotton genome sequence near the arbitrarily assigned 5' end of the partial crylA coding sequence inserted into the genome of cotton event 531.
  • SEQ ID NO:26 corresponds to Primer J, a sequence which is or is complementary to a part of the 3' end flanking cotton genome sequence near the arbitrarily assigned 3' end of the full length functional inserted DNA in cotton event 531.
  • SEQ ID NO:27 corresponds to Primer K, a sequence which is or is complementary to a sequence within the 5' flanking cotton genome sequence near the arbitrarily assigned 5' end of the partial 7S 3' sequence present in event 531.
  • SEQ ID NO:28 corresponds to Primer L, a sequence which is or is complementary to a sequence within the 3' flanking cotton genome sequence near the arbitrarily assigned 3' end of the partial 7S 3' sequence present in event 531.
  • SEQ ID NO:29 corresponds to an amplicon sequence produced using primers corresponding to SEQ ID NO:27 and SEQ ID NO:28 together with native cotton template DNA.
  • SEQ ID NO:30 corresponds to an amplicon sequence produced using primers corresponding to SEQ ID NO:25 and SEQ ID NO:26 together with native cotton template DNA.
  • SEQ ID NO:31 corresponds to the nucleotide sequence of a part of the cotton genome flanking the arbitrarily assigned 5' end of the partial 7S 3' DNA sequence inserted into the cotton genome in event 531.
  • SEQ ID NO:32 corresponds to a sequence which is diagnostic for the presence of event 531 DNA in a biological sample, nucleotides 1-10 corresponding to the first 10 cotton genome nucleotides adjacent to and flanking the arbitrarily assigned 5' end of the partial 7S 3' sequence inserted into the genome in event 531, nucleotides 11-20 corresponding to the first ten nucleotides within the arbitrarily assigned 5' end of the partial 7S 3' sequence inserted into the genome in event 531.
  • SEQ ID NO:33 corresponds to the partial 7S 3' sequence inserted present in event 531, along with a part of the 5' and 3' cotton genome flanking sequences.
  • biological sample is intended to include nucleic acids, polynucleotides, DNA, RNA, tRNA, cDNA, and the like in a composition or fixed to a substrate which enables the sample to be subjected to molecular probe analysis or thermal amplification using oligonucleotide probes and/or primers.
  • the term “cotton” means Gossypium hirsutum and includes all plant varieties that can be bred with cotton, including wild cotton species.
  • a transgenic "event” is produced by transformation of plant cells with heterologous DNA, i.e., a nucleic acid construct that includes a transgene of interest, regeneration of a population of plants resulting from the insertion of the transgene into the genome of the plant, and selection of a particular plant characterized by insertion into a particular genome location.
  • the term “event” refers to the original transformant and progeny of the transformant that include the heterologous DNA.
  • the term “event” also refers to progeny produced by a sexual outcross between the transformant and another variety that include the heterologous DNA.
  • vent also refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA.
  • transgenic plants can also be mated to produce offspring that contain two independently segregating added, exogenous genes. Selfing of appropriate progeny can produce plants that are homozygous for both added, exogenous genes.
  • Back-crossing to a parental plant and out-crossing with a non-transgenic plant are also contemplated, as is vegetative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several references, e.g., Fehr, in Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison WI (1987).
  • Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar, inbred line, or elite ge ⁇ nplasm which is the recurrent parent.
  • the source of the trait to be transferred is called the donor parent.
  • the resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
  • individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent.
  • the resulting parent is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.
  • a “probe” is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, e.g., a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA from cotton event 531 whether from a cotton plant or from a sample that includes DNA from the event. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
  • Primer pairs of the present invention refer to their use for amplification of a target nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
  • PCR polymerase chain reaction
  • Probes and primers are generally about 11 nucleotides or more in length, preferably 18 nucleotides or more, more preferably 24 nucleotides or more, and most preferably 30 nucleotides or more. Such probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions. Preferably, probes and primers according to the present invention have complete sequence similarity with the target sequence, although probes differing from the target sequence and that retain the ability to hybridize to target sequences may be designed by conventional methods. Primers and probes are often interchangeable, and so primers may be used as probes and probes may be used as primers where effective.
  • Thermal amplification primers can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • Primers and probes based on the flanking DNA and inserted heterologous sequences disclosed herein can be used to confirm (and, if necessary, to correct) the disclosed sequences by conventional methods, e.g., by re-cloning and sequencing such flanking DNA and inserted sequences.
  • nucleic acid probes and primers of the present invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from a transgenic event in a sample.
  • Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. As used herein, two nucleic acid molecules are said to be capable of specifically hybridizing to one another if the two molecules are capable of forming an anti-parallel, double-stranded nucleic acid structure.
  • a nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if they exhibit complete complementarity.
  • molecules are said to exhibit "complete complementarity" when every nucleotide of one of the molecules is complementary to a nucleotide of the other.
  • Two molecules are said to be “minimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional "low-stringency” conditions.
  • the molecules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional "high-stringency” conditions.
  • a nucleic acid molecule In order for a nucleic acid molecule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and salt concentrations employed.
  • a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complement of the nucleic acid sequence to which it is being compared under high stringency conditions.
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C.
  • a nucleic acid of the present invention will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:32 or complements thereof or fragments of either under moderately stringent conditions, for example at about 2.0 x SSC and about 65°C.
  • a nucleic acid of the present invention will specifically hybridize to one or more of the nucleic acid molecules set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:32 or complements or fragments of either under high stringency conditions.
  • a preferred marker nucleic acid molecule of the present invention has the nucleic acid sequence set forth SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO:21 or complements thereof or fragments of either.
  • a preferred diagnostic marker nucleic acid molecule of the present invention shares between from about 80% to about 100% or from about 90% to about 100% sequence identity with the nucleic acid sequence set forth in SEQ ID NO:l, 2, 3, SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:21 or complement thereof or fragments of either.
  • a preferred marker nucleic acid molecule of the present invention shares between from about 95% to about 100% sequence identity with the sequence set forth in SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, and SEQ ID NO:21 or complements thereof or fragments of either.
  • SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:21 and complements thereof may be used as markers in plant breeding methods to identify the progeny of genetic crosses similar to the methods described for simple sequence repeat DNA marker analysis, in "DNA markers: Protocols, applications, and overviews: (1997) 173-185, Cregan, et al., eds., Wiley-Liss NY; all of which is herein incorporated by reference in its' entirely.
  • the hybridization of the probe to the target DNA molecule can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody based tags, and chemiluminescent tags.
  • stringent conditions are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complement) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
  • specific for (a target sequence) indicates that a probe or primer hybridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
  • an "isolated" nucleic acid is one that has been substantially separated or purified away from other nucleic acid sequences in the cell of the organism in which the nucleic acid naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, by conventional nucleic acid-purification methods.
  • the term also embraces recombinant nucleic acids and chemically synthesized nucleic acids.
  • transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism such as a host plant, resulting in genetically stable inheritance.
  • Host plants containing the transformed nucleic acid fragments are referred to as "transgenic plants”.
  • the term "diagnostic” refers to the fact that, for the purposes of identifying nucleic acid sequences as those contained within or derived from cotton event 531, any one or more of the novel DNA sequences set forth herein comprise the cotton genome flanking sequences adjacent to and linked to the arbitrarily assigned ends of the inserted heterologous DNA sequences are necessary and sufficient as being descriptive as a distinguishing characteristic of the event 531 genome, so long as the sequence comprises at least a part of one of the ends of the inserted heterologous DNA sequence or the cotton genome sequence flanking or adjacent to one of these ends and includes at least the two nucleotides, the di-nucleotide, comprising the point at which the cotton genome sequence and the inserted heterologous DNA sequence are linked together by a phosphodiester bond.
  • amplified DNA refers to the product of nucleic-acid amplification of a target nucleic acid sequence that is part of a nucleic acid template.
  • DNA extracted from a cotton plant tissue sample may be subjected to nucleic acid amplification method using a primer pair that includes a first primer derived from sequences in the genome of the plant which are adjacent to one end of the inserted heterologous DNA sequence, and a second primer derived from sequences within the inserted heterologous DNA sequence, to produce an amplicon that is diagnostic for the presence of the event DNA.
  • the amplicon is of a length and has a sequence that is also diagnostic for the event.
  • the amplicon contains at least the dinucleotide sequence comprising the two nucleotides forming the link between one end of the inserted heterologous DNA and the first nucleotide within the native genome DNA sequence, which is immediately adjacent to the end of the inserted heterologous DNA sequence as well as the combined sequences of the first and the second primers.
  • the amplicon may range in length from about five hundred nucleotide base pairs, to about three hundred nucleotide base pairs, to about two hundred nucleotide base pairs, to about fifty nucleotide base pairs, to about the combined length of the primer pairs plus one nucleotide base pair.
  • a primer pair can be derived from flanking sequence within the cotton plant genome sequences linked to both ends of the inserted heterologous DNA sequence so as to produce an amplicon that includes the entire insert nucleotide sequence.
  • a member of a primer pair which is derived from the plant genomic sequence may be located a distance from either end of the inserted DNA sequence, and this distance can range from one nucleotide base pair up to about twenty thousand nucleotide base pairs.
  • sequences which are diagnostic for the event selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:14, SEQ ID NO: 15, SEQ ID NO:16, and SEQ ID NO:21.
  • the use of the term "amplicon” specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
  • Nucleic-acid amplification can be accomplished by any of the various nucleic-acid amplification methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Patent Nos.
  • the amplicon produced by these methods may be detected by a plurality of techniques.
  • One such method is Genetic Bit Analysis (Nikiforov, et al. Nucleic Acid Res. 22:4167-4175, 1994) in which an oligonucleotide is designed which overlaps both the adjacent flanking genomic UNA sequence and the inserted DNA sequence.
  • the oligonucleotide is immobilized in wells of a microwell plate.
  • a single-stranded amplicon can be used to hybridize to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labelled ddNTPs specific for the expected next base, as determined by fluorescent or immunological based detection methods.
  • a positive signal indicates the presence of the insert/flanking sequence in the sample and is diagnostic for the presence of the event 531 nucleic acid.
  • Another method for detecting the amplicon diagnostic for the event 531 nucleic acid in a sample is the Pyrosequencing technique as described by Winge (Innov. Pharma. Tech. 00:18-24, 2000). In this method an oligonucleotide is designed that overlaps the adjacent genomic DNA and insert DNA junction.
  • the oligonucleotide is hybridized to a single-stranded amplicon from the region of interest (amplicon produced using a first primer complimentary to a sequence within the inserted heterologous DNA sequence and a second primer complimentary to a sequence within the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5' phosphosulfate and luciferin.
  • dNTPs are added individually and incorporation results in the production of photons of light which are detected and measured, and which is diagnostic for the event 531 nucleic acid sequence in a sample.
  • Fluorescence Polarization as described by Chen, et al., (Genome Res. 9:492-498, 1999) is a method useful for detecting the diagnostic amplicon of the present invention. Using this method, an oligonucleotide is designed which overlaps the junction of the genomic flanking sequence and inserted DNA sequence.
  • the oligonucleotide is hybridized to a single-stranded thermal amplification product from the region of interest (using a first primer complementary to a part of the inserted heterologous DNA sequence and a second primer complimentary to a part of the genomic DNA sequence flanking the proximal terminal end of the inserted heterologous DNA sequence) and incubated in the presence of a DNA polymerase and a ddNTP labeled with a fluorophore which emits a particular wavelength of light (emission spectrum) upon excitation with light of a wavelength different from the emission spectrum (excitation spectrum).
  • Single base extension results in incorporation of the fluorphore labeled ddNTP.
  • Incorporation can be measured as a change in fluorescence polarization using a fluorimeter.
  • a change in fluorescence polarization indicates the presence of the transgene insert/flanking sequence within the amplicon due to successful amplification, hybridization, and single base extension, and is diagnostic for the event 531 nucleic acid in a sample.
  • Taqman ® PE Applied Biosystems, Foster City, CA
  • FRET fluorescence resonance emissions tagged oligonucleotide probe
  • the FRET probe and thermal amplification primers (a first primer complementary to a part of the inserted heterologous DNA sequence and a second primer complementary to a part of the adjacent or flanking cotton genomic DNA sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe.
  • a fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization, and is diagnostic for the presence of the event 531 nucleotide sequence in a sample.
  • Molecular Beacons have been described for use in sequence detection as described in Tyangi, et al. (Nature Biotech.14:303-308, 1996) Briefly, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in a probe exhibiting a secondary structure that maintains the fluorescent and quenching moieties in close proximity.
  • the FRET probe and thermal amplification primers are cycled in the presence of a thermostable polymerase and dNTPs.
  • hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties.
  • a fluorescent signal results.
  • a fluorescent signal indicates the presence of the flanking/transgene insert sequence due to successful amplification and hybridization, and is diagnostic for the event 531 nucleic acid in a sample.
  • Ligase chain reaction is also contemplated as being diagnostic for the event 531 nucleic acids in a sample. All of the above methods can be modified to determine the zygosity of a particular sample of nucleic acids derived from a single source.
  • a cotton event 531 plant which is homozygous for the event 531 allele contains within its genome two copies of the event 531 allele characteristic of and diagnostic for the cotton event 531 genome, and thus when selfed would breed true.
  • a cotton event 531 homozygous plant can be crossed with another variety of cotton, and the result of that cross would be plants that were heterozygous for the event 531 allele.
  • Methods are envisioned in which one skilled in the art could determine the zygosity of a particular plant with reference to the event 531 allele. This method requires at least three oligonucleotide sequences as set forth herein.
  • a heterozygosity assay comprising a thermal amplification reaction comprising event 531 nucleic acid sequences in a sample as the template and Primer K (SEQ ID NO: 27) and Primer L (SEQ ID NO: 28) described herein would produce an amplicon of about 443 base pairs in length, which is diagnostic for the presence of event 531 DNA in a sample.
  • the same primers would also produce an amplicon of about 209 base pairs in length, which is diagnostic for the presence of cotton DNA other than event 531 in a sample. Therefore, in a biological sample comprising cotton genomic DNA, the production of only the larger of the two sequences would indicate that the cotton plant source genome would contain only event 531 DNA.
  • cotton event 531 contains a primary functional insert containing a significant portion of the transformation plasmid, PV-GHBK04.
  • a second nonfunctional insertion includes a right border initiation event that continues up to and is linked with the 7S 3' transcriptional termination sequence and 3' of the CrylA coding sequence within the primary functional insertion.
  • a third insertion which is unlinked to the first two, i.e. does not segregate true with the first and second inserted DNA segments, consists of a part of the 7S 3' plant transcriptional termination and polyadenylation sequence.
  • These three segments are detectable and diagnostic for the event 531 nucleic acid sequences in a sample, in particular in plants which have been selfed since the origination of the 531 event.
  • the third segment fails to breed true and so may not produce amplicons which, as taught herein, are diagnostic for the 531 event. It should be noted that the absence of particular amplicons associated with this particular segment should not be diagnostic of a cotton plant other than the cotton plant event 531 in a sample.
  • the inventors herein describe the molecular analyses that have been performed on transgenic cotton event 531 to further define the ends of the T-DNA insertions and identify the cotton genomic DNA flanking the T-DNA insertions. Genome walking studies combined with nucleotide sequencing has resulted in the identification of the DNA sequences at the arbitrarily assigned 5' and 3' ends of the primary functional insert, as well as cotton genomic DNA flanking the 5' and 3' ends of the T-DNA insertions in the transgenic cotton event 531.
  • the second, nonfunctional, T-DNA insertion, containing a portion of the cry I A coding region, is located at the arbitrarily assigned 5'end (7S 3' portion) of the primary insert.
  • the inventors therefore disclose herein the analysis of the genome architecture of the inserted sequence and flanking cotton genomic DNA sequences in transgenic cotton event 531 including about 309 nucleotides of cotton genomic DNA flanking the arbitrarily assigned 5'end of the insertion and about 211 base pairs of cotton genomic DNA flanking the arbitrarily assigned 3 'end of the insertion event in cotton event 531.
  • a second inserted sequence containing a part of the 3' coding region for a CrylA nucleic acid sequence is present in proximity to the arbitrarily assigned 5 '-end (7S 3' portion) of the primary insert, thus defining a complex arrangement of these genetic elements derived from the transformation plasmid PV- GHBK04 at a single point of insertion in the genome of the transgenic cotton event 531, notwithstanding the third sequence comprising the portion of a 7S 3' end sequence at an unlinked single point of insertion in the cotton genome.
  • the second inserted sequence is effectively an inverted repeat of the terminal coding sequence for the Cryl Ac protein and associated 7S 3' termination sequence within the full length primary functional inserted DNA sequence in event 531.
  • the physical organization of the inserted sequences is set forth in Figure 1 herein.
  • a method for producing a cotton plant that is resistant to lepidopteran insect infestation may be conducted with the following steps: 1) sexually crossing a first cotton plant grown from the cotton seed event 531 comprising a DNA molecule selected from the group consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:21 that confers resistance to lepidopteran insect infestation, and a second cotton plant that lacks the resistance to lepidopteran insect infestation, thereby producing a plurality of first progeny plants;
  • the first progeny plant that is resistant to lepidopteran insect infestation or the second progeny plant that is resistant to lepidopteran insect infestation may be backcrossed to the second cotton plant or a third cotton plant and a cotton plant that is resistant to lepidopteran insect damage infestation be produced.
  • DNA detection kits can be developed using the compositions disclosed herein and the methods well known in the art of DNA detection.
  • the kits are useful for identification of cotton event 531 DNA in a sample and can be applied to methods for breeding cotton plants containing 531 DNA.
  • the kits contain one or more DNA sequences comprising at least 11 contiguous nucleotides homologous or complementary to sequences selected from the group consisting of SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO:l l, SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, S
  • PV-GHBK04 in cotton event 531 provides novel nucleic acid sequences at the 5' and 3' cotton genome/insert junctions at each of three sites of insertions. These novel nucleic acid sequences are useful for detecting DNA from cotton event 531 in a sample using various methods well known in the art. The following provides non-limiting examples of how a skilled artisan might use these novel nucleic acid sequences to detect cotton event 531 in a sample.
  • Example 1 Cotton Genomic DNA Isolation.
  • DNA from cotton event 531 was extracted from seed tissue.
  • DNA was extracted from both seed and leaf tissues from the control substance (non-transgenic cotton seed and leaf tissue).
  • DNA from seed was isolated by processing the seed to a fine powder using a commercially available blender. Approximately 2 grams of the processed seed was transferred to a 50 ml conical tube, and -16 ml of CTAB extraction buffer [1.5% (w:w) CTAB, 75 mM Tris-HCl pH 8.0, 100 mM EDTA pH 8.0, 1.05 M NaCl, and 0.75% (w:w) PVP (MW 40,000)] was added to the processed seed. The samples were incubated at 65°C for approximately 30 minutes with intermittent mixing and then allowed to cool to room temperature.
  • the aqueous (upper) phase was removed, mixed with an equal volume (-15 ml) of CTAB precipitation buffer [1% (w:w) CTAB, 50 mM Tris pH 8.0, and 10 mM EDTA pH 8.0] and allowed to stand at room temperature for approximately 1 hour.
  • CTAB precipitation buffer [1% (w:w) CTAB, 50 mM Tris pH 8.0, and 10 mM EDTA pH 8.0] and allowed to stand at room temperature for approximately 1 hour.
  • the samples were centrifuged at -10,000 x g to pellet the DNA, the supernatant was decanted, and the pellet was dissolved in approximately 2 ml of high salt TE [lOmM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, and 1 M NaCl] by incubating at 37°C with gentle swirling for approximately 2 hours. Centrifugation was performed at -23,000 x g to pellet any remaining impurities.
  • the supernatant was removed, placed into a clean 15 ml tube, and approximately 1/10 volume (-150 ⁇ l) of 3M NaOAc, pH 5.2, and 2 volumes (-4 ml relative to the supernatant) of chilled 100% ethanol were added to precipitate the DNA.
  • the precipitated DNA was spooled into a microfuge tube containing approximately 1 ml of 70% ethanol.
  • the DNA was pelleted in a microfuge at maximum speed (14,000 rpm) for 5 minutes, dried, and re-dissolved in TE, pH 8.0 in a 4°C refrigerator overnight.
  • the non-transgenic cotton genomic DNA used as a control was isolated from leaf tissue that was frozen in liquid nitrogen and ground into a fine powder using a mortar and pestle. Approximately 1 g of the ground leaf tissue was transferred to a 13 ml centrifuge tube and 6 ml of extraction buffer [2.5 ml DNA extraction buffer (350 mM sorbitol, 100 mM Tris pH 7.5, 5 mM EDTA, 0.38% (w/v) sodium bisulfite), 2.5 ml nuclei lysis buffer (200 mM Tris pH 7.5, 50 mM EDTA, 2 M NaCl, 2% (w/v) CTAB), and 1 ml Sarkosyl (5% (w/v) solution)] was added.
  • 2.5 ml DNA extraction buffer 350 mM sorbitol, 100 mM Tris pH 7.5, 5 mM EDTA, 0.38% (w/v) sodium bisulfite
  • 2.5 ml nuclei lysis buffer 200 mM
  • the samples were incubated at 65 °C for approximately 30 minutes with intermittent mixing.
  • Four and a half milliliters of chloroform:isoamyl alcohol (24:1 (v/v)) at room temperature was added to the samples.
  • the suspension was mixed for 2 to 3 minutes, and the two phases separated by centrifugation for 15 minutes at -2,000 x g at 4°C.
  • the aqueous (top) layer was removed using a transfer pipet and placed into a 13 ml centrifuge tube.
  • Five milliliters of 100%) isopropanol were added, and the tubes were mixed by inversion to precipitate the DNA.
  • the precipitated DNA was spooled into a microfuge tube containing 500 1 of 70% ethanol.
  • the DNA was pelleted in a microfuge at maximum speed (14,000 rpm) for 2 minutes.
  • the DNA was dried and dissolved in TE buffer in a 4°C refrigerator overnight.
  • PCR-based Universal Genome Walker KitTM as per the manufacturer's protocol followed by nucleotide sequencing of the PCR products.
  • PCR assays were developed using one primer complementary to cotton genomic DNA and another primer complementary to inserted transgene DNA.
  • Primer D SEQ ID NO:20
  • Primer C SEQ ID NO: 19
  • Primer A SEQ ID NO: 17
  • Primer B SEQ ID NO: 18
  • the PCR assays were performed using 10 - 100 ng of cotton event 531 genomic DNA template in a 50 ⁇ l reaction volume containing a final concentration of 1.1 mM Mg 2+ , 0.4 ⁇ M of each primer, 200 ⁇ M each dNTP, and 2.5 units of Taq DNA polymerase.
  • the reactions for the PCR assays were performed under the following cycling conditions: 1 cycle at 94°C for 3 minutes; 38 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 90 seconds; 1 cycle at 72°C for 10 minutes.
  • the PCR products were separated using agarose gel electrophoresis, visualized by ethidium bromide staining, excised from the gel, and subjected to DNA sequencing using dye-terminator chemistry to confirm the sequences.
  • the cotton event 531 samples generated the expected size PCR products of about 1411 bp (SEQ ID NO: 15) for the 5' flanking sequence and about 589 bp (SEQ ID NO:14) for the 3' flanking sequence. Therefore, the novel nucleic acid sequences at the junction of inserted DNA and cotton genomic DNA in cotton event 531 are useful for detecting DNA derived from cotton event 531 in a sample.
  • the amplicon products were sequenced to determine the precise sequence of the flanking sequence-insert junctions.
  • sequence of SEQ ID NO: 15 is comprised of a part of the 5' end flanking cotton genome sequence (SEQ ID NO: 6) upstream or 5' to the 5' end of the partial crylAc coding sequence fragment, SEQ ID NO: 6 being physically linked at its 3' end to the arbitrarily assigned 5' end of the inserted partial crylAc coding sequence (SEQ ID NO: 5).
  • SEQ ID NO: 6 The last ten nucleotides set forth in SEQ ID NO:6 and the first ten nucleotides set forth in SEQ ID NO:5 correspond to SEQ ID NO:2 which is a sequence diagnostic for the event 531 DNA in a sample.
  • sequence of at least eleven nucleotides in length selected from the sequences in SEQ ID NO: 15 which comprises at least the di-nucleotide in SEQ ID NO:2 from position 10 through position 11 and complements thereof are diagnostic for the cotton event 531 nucleic acid sequences in a sample.
  • sequence of SEQ ID NO: 14 is comprised of a part of the 3' end flanking cotton genome sequence (SEQ ID NO:l 1) downstream or 3' to the arbitrarily assigned 3' end of the primary full length and functional inserted DNA sequence in event 531, SEQ ID NO: 11 being physically linked at its 5' end to the arbitrarily assigned 3' end of the primary full length and functional inserted DNA sequence in event 531 (SEQ ID NO: 10).
  • the last ten nucleotides of SEQ ID NO:10 and the first ten nucleotides of SEQ ID NO:ll comprise a 20mer nucleotide sequence set forth in SEQ ID NO:l which is diagnostic for the event 531 DNA in a sample.
  • a sequence of at least eleven nucleotides in length selected from the sequences in SEQ ID NO: 14 which comprises at least the di-nucleotide in SEQ ID NO:l from position 10 through position 11, and complements thereof are diagnostic for the cotton event 531 nucleic acid sequences in a biological sample.
  • Thermal amplification assays were also developed to determine the 5' end flanking sequence corresponding to the termination sequence-genome junction within the full length functional inserted sequence.
  • the DNA sequence within the termination sequence of the 7S 3' termination sequence linked to the full length crylAc coding sequence inserted into Coker 312 resulting in the event 531 genome was determined, along with sequence beyond the termination sequence and out into the flanking sequence region.
  • the resulting sequence consists of the sequence set forth in SEQ ID NO:16, and is comprised of sequences set forth in SEQ ID NO:7 (flanking cotton genome sequence 5' to, upstream of, or adjacent to the inserted DNA sequence) and SEQ ID NO: 8 (7S 3' inserted DNA sequence within the primary full length and functional inserted DNA sequence).
  • SEQ ID NO:3 comprising the junction of SEQ ID NO:7 and SEQ ID NO:8 is diagnostic for the event 531.
  • a sequence of at least eleven nucleotides in length selected from the sequences in SEQ ID NO: 16 which comprises at least the di-nucleotide in SEQ ID NO:3 from position 10 through position 11, and complements thereof, are diagnostic for the cotton event 531 nucleic acid sequences in a biological sample.
  • Primers E (SEQ ID NO:21) and F (SEQ ID NO:22) were designed to amplify a sequence comprising the arbitrarily assigned 5' end of the partial 7S 3' sequence inserted into the cotton genome linked to the cotton flanking genome sequence 5' to the inserted sequence.
  • An amplicon comprising a diagnostic sequence as set forth in SEQ ID NO:32 is obtained when these primers, as a primer pair or primer set, are used in a thermal amplification reaction along with cotton event 531 template DNA in a sample.
  • a sequence comprising at least eleven nucleotides in length selected from the sequences as set forth in SEQ ID NO:32 and which contain at least the di-nucleotide in SEQ ID NO:32 from position 10 through position 11, and complements thereof, are diagnostic for the cotton event 531 nucleic acid sequences in a biological sample.
  • Primers G (SEQ ID NO:23) and H (SEQ ID NO:24) were designed to amplify a sequence comprising the arbitrarily assigned 3' end of the partial 7S 3' sequence inserted into the cotton genome linked to the cotton flanking genome sequence 3' to the inserted sequence.
  • the 5' and 3' genomic DNA sequences flanking the second insert containing a portion of the 7S 3' genetic element were identified using one primer designed to the 5' or 3' genomic DNA sequence flanking the insertion (Primers F and H, respectively), paired with a second primer in the insertion (Primers E and G, respectively).
  • the PCR analyses were conducted using 100 ng of genomic DNA template in a 50 ⁇ l reaction volume containing a final concentration of 1.5 mM Mg 2+ , 0.2 ⁇ M of each primer, 200 ⁇ M each dNTP, and 1 unit of Taq DNA polymerase.
  • the reactions were performed under the following cycling conditions: 1 cycle at 94°C for 3 minutes; 38 cycles of 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1.5 minutes; 1 cycle at 72°C for 10 minutes.
  • the PCR products were separated on 1.0 % agarose gels and visualized by ethidium bromide staining.
  • the PCR products generated from cotton event 531 DNA were excised from the gel, and subjected to DNA sequencing using dye-terminator chemistry to confirm the sequences.
  • An amplicon comprising a diagnostic sequence as set forth in SEQ ID NO:4 is obtained when these primers, as a primer pair or primer set, are use in a thermal amplification reaction along with cotton event 531 template DNA in a sample.
  • a sequence comprising at least eleven nucleotides in length selected from the sequences as set forth in SEQ ID NO:4 and which contain at least the di-nucleotide in SEQ ID NO:4 from position 10 through position 11, and complements thereof, are diagnostic for the cotton event 531 nucleic acid sequences in a biological sample.
  • Example 3 Identification of the native cotton genome sequences into which heterologous DNA sequences were inserted to form cotton event 531 and heterozygosity assays developed therefrom.
  • Amplicons useful for determining the heterozygosity or homozygosity of the cotton genome with reference to event 531 are required in order to determine conclusively whether a particular line of cotton comprises event 531 sequences or otherwise.
  • Primers for use in detecting the native cotton genomic DNA sequence in a sample were designed which, when used with a template cotton genome DNA comprising DNA derived from other than an event 531 source, produce an amplicon which is diagnostic for at least one allele present in the template which is representative of native cotton genomic DNA uninterrupted by the inserted DNA found in event 531.
  • a primer pair consisting of Primer I (SEQ ID NO:25) which is or is complementary to cotton genome sequences within a part of the genome flanking the arbitrarily assigned 5' end of the partial crylAc coding sequence in event 531, and Primer J (SEQ ID NO:26) which is or is complementary to cotton genome sequences within a part of the genome flanking the arbitrarily assigned 3' end of the primary full length and functional inserted DNA in event 531, when used together in a thermal amplification reaction with cotton template DNA other than event 531 DNA produce an amplicon comprising 374 base pairs in length, one strand of which corresponds to the sequence set forth in SEQ ID NO:30, and which is diagnostic for the presence of a DNA sequence in a biological sample derived from a cotton genome other than event 531.
  • PCR analyses of the functional insert site were performed using a primer specific to the genomic DNA sequence flanking the 5 '-end of the insertion in the forward direction (Primer I), paired with a second primer specific to the genomic DNA sequence flanking the 3 '-end of the insertion (Primer J).
  • the PCR analyses were conducted using 20 ng of genomic DNA template in a 50 ⁇ l reaction volume containing a final concentration of 1.5 mM Mg 2+ , 0.4 ⁇ M of each primer, 200 ⁇ M each dNTP, and 2.5 units of Taq DNA polymerase.
  • the reactions were performed under the following cycling conditions: 94°C for 3 minutes; 38 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1.0 minute; 1 cycle at 72°C for 10 minutes.
  • the PCR products were separated on a 1.5-2.0 % agarose gel and visualized by ethidium bromide staining. Following electrophoresis, PCR products generated from Coker 312 non-transgenic DNA were excised from the gel and sequenced using dye-terminator chemistry.
  • Primers for use in detecting the native cotton genomic DNA sequence in a sample were designed which, when used with a template cotton genome DNA comprising DNA derived form other than an event 531 source, produce an amplicon which is diagnostic for at least one allele present in the template which is representative of native cotton genomic DNA uninterrupted by the inserted DNA found in event 531.
  • a primer pair consisting of Primer K (SEQ ID NO: 27) which is or is complementary to cotton genome sequence within a part of the genome flanking the arbitrarily assigned 5' end of the partial 7S 3' sequence inserted in event 531, and Primer L (SEQ ID NO:28) which is or is complementary to cotton genome sequences within a part of the genome flanking the arbitrarily assigned 3' end of the partial 7S 3' sequence inserted into event 531, when used together in a thermal amplification reaction with cotton template DNA other than event 531 DNA produce an amplicon comprising 209 base pairs in length, one strand of which corresponds to the sequence set forth in SEQ ID NO:29, and which is diagnostic for the presence of a DNA sequence in a biological sample derived from a cotton genome other than event 531.
  • Primer K SEQ ID NO: 27
  • Primer L SEQ ID NO:28
  • An amplicon spanning the insertion site of the partial 7S 3' sequence was generated from Coker 312 non-transgenic DNA.
  • the insertion site was amplified from Coker 312 using one primer specific to the genomic DNA sequence identified 5' of the insert DNA (Primer K) and a second primer specific to the genomic DNA sequence identified 3' of the inserted DNA (Primer L).
  • the PCR analyses were conducted using 20 ng of genomic DNA template in a 50 ⁇ l reaction volume containing a final concentration of 1.5 mM Mg , 0.4 ⁇ M of each primer, 200 ⁇ M each dNTP, and 2.5 units of Taq DNA polymerase.
  • the reactions were performed under the following cycling conditions: 94°C for 3 minutes; 38 cycles at 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 1.0 minutes; 1 cycle at 72°C for 10 minutes.
  • the PCR products were separated on a 2.0 % agarose gel and visualized by ethidium bromide staining. Following electrophoresis, PCR products generated from Coker 312 non-transgenic DNA were excised from the gel and sequenced using dye-terminator chemistry.

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Abstract

La présente invention concerne des analyses servant à détecter la présence de séquences d'acide nucléique d'évènement du coton 531 dans un échantillon biologique au moyen d'une séquence d'ADN d'un gène hybride recombiné inséré dans le génome du coton et de séquences génomiques flanquant le site d'insertion dans un génome de coton.
PCT/US2001/043297 2000-11-20 2001-11-20 Evenement du coton pv-ghbk04 (531) et compositions et procedes permettant de detecter la presence de ce dernier WO2002040677A2 (fr)

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AU2002236442A AU2002236442A1 (en) 2000-11-20 2001-11-20 Cotton event pv-ghbk04 (531) and compositions and methods for detection thereof
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