Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-13
2004-03-30
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S009000, C536S023100, C536S024300
Reexamination Certificate
active
06713259
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the field of plant molecular biology, more specifically to the invention of a DNA construct for conferring improved insect resistance to a corn plant, and even more specifically to an insect resistant corn plant transformation event MON810 and progeny thereof, and to assays for detecting the presence of MON810 DNA in a sample and compositions thereof.
BACKGROUND OF THE INVENTION
As used herein, the term “event” refers to a plant produced by transformation and regeneration of a single plant cell with heterologous DNA, i.e., a DNA construct that includes a transgene of interest. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome each of a plant cell. Based on the expression of the transgene or other desirable characteristics, a particular event is selected.
The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proximity of transcriptional regulation elements (e.g., enhancers) close to the integration site (Weising et al., 1988, “Foreign Genes in Plants,”
Ann. Rev. Genet.
22:421-477). For this reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of an introduced gene of interest. For example, it has been observed in plants and in other organisms that there may be wide variations in levels of expression of a heterologous gene introduced into the chromosome of a plants' genome among individually selected events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. For this reason, it is common to produce hundreds to thousands of different events and screen those events for a single event that has desired transgene expression levels and patterns for commercial purposes. An event that has desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions.
It would be advantageous to be able to detect the presence of a particular event in order to determine whether progeny of a sexual cross contain a transgene of interest. In addition, a method for detecting a particular event would be helpful for complying with regulations requiring the pre-market approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well known nucleic acid detection method including but not limited to thermal amplification (PCR™) or DNA hybridization using nucleic acid probes. Typically, for the sake of simplicity and uniformity of reagents and methodologies for use in detecting a particular DNA construct that has been used for transforming various plant varieties, these detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc., because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between constructs that differ only with reference to the coding sequence. In addition, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted heterologous DNA (“flanking DNA”) is known. An event-specific thermal amplification (PCR™) assay is discussed, for example, by Windels et al. (
Med. Fac. Landbouww, Univ. Gent
64/5b:459-462, 1999), who identified glyphosate tolerant soybean event 40-3-2 using a thermal amplification primer set spanning the junction between the insert and flanking DNA. Specifically, one primer was comprised of sequence from within the insert and a second primer was comprised of sequence from flanking DNA.
The inventor herein discloses novel and useful isolated nucleic acid sequences, as well as methods for detecting these nucleic acids in a biological sample, and kits comprising the reagents necessary for use in detecting these nucleic acids in a biological sample.
SUMMARY OF THE INVENTION
The inventor herein has discovered a corn event, MON810, that is resistant to Lepidopteran insect infestation, and also provides compositions and methods for detecting the presence of genomic DNA from this event in a biological sample.
According to one aspect of the invention, DNA sequences are provided that comprise at least one junction sequence of corn event MON810 selected from the group consisting of 5′-ACATCCTTTGCCATTGCCCA-3′ (SEQ ID NO:1) and 5′-GAACGAGGACTTTCGGTAGC-3′ (SEQ ID NO:2) and complements thereof (i.e., one or both junction sequences), wherein a junction sequence spans the junction between heterologous DNA inserted into the corn genome and DNA from the corn genome flanking the insertion site and is diagnostic for the event. Included are DNA sequences that comprise at least 10 or more (e.g., 15, 25, 50) nucleotides of insert sequence from corn event MON810 and similar length of flanking DNA from corn event MON810. Also included are DNA sequences which comprise 15 or more nucleotides of contiguous insert sequence from corn event MON810 and at least one nucleotide of flanking DNA from corn event MON810 adjacent to the insert sequence. Such DNA sequences are diagnostic for corn event MON810. Nucleic acid amplification of genomic DNA from the event produces an amplicon comprising such diagnostic DNA sequences.
According to another aspect of the invention, corn plants comprising such DNA sequences and seed from such corn plants are provided.
According to another aspect of the invention, flanking sequence primers for detecting corn event MON810 are provided. Such flanking sequence primers comprise an isolated nucleic acid sequence comprising at least 15 contiguous nucleotides from nucleotides 1-244 of SEQ ID NO:3 (arbitrarily designated herein as the 5′ flanking sequence), at least 15 contiguous nucleotides from nucleotides 274-879 of SEQ ID NO:4 (arbitrarily designated herein as the 3′ flanking sequence), or the complements thereof.
According to another aspect of the invention, primer sets that are useful for nucleic acid amplification, for example, are provided. Such primer sets comprise a primer comprising a nucleotide sequence of at least 10-15 contiguous nucleotides in length which is or is complementary to one of the above-described genomic flanking sequences (SEQ ID NO:3 or SEQ ID NO:4) and a second primer comprising at least an isolated 10-15 contiguous nucleotides of heterologous DNA inserted into the plant DNA sequence, i.e., the DNA sequence inserted into the recombinant corn plant event MON810. The second primer is preferably a polynucleotide sequence which is or is complementary to the insert sequence adjacent to the plant genomic flanking DNA sequence as set forth in SEQ ID NO:3 from nucleotide position 245 through 566 and in SEQ ID NO:4 from nucleotide position 1 through 273. Of course, it is well within the skill in the art to obtain additional sequence further out into the genome sequence flanking either end of the inserted heterologous DNA sequence for use as a primer sequence that can be used in such primer pairs for amplifying the sequences that are diagnostic for the MON810 corn event. For the purposes of this disclosure, the phrase “further out into the genome sequence flanking either end of the inserted heterologous DNA sequence” refers specifically
Ball Timothy K.
Hoerner Dennis R.
Monsanto Technology LLC
Myers Carla J.
Sakelaris Sally
LandOfFree
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