Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-11
2002-05-28
Jones, W. Gary (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S023100, C536S024300
Reexamination Certificate
active
06395485
ABSTRACT:
BACKGROUND OF THE INVENTION
The phenotypic expression of a transgene in a plant is determined both by the structure of the gene itself and by its location in the plant genome. At the same time the presence of the transgene (in a foreign DNA) at different locations in the genome will influence the overall phenotype of the plant in different ways. The agronomically or industrially successful introduction of a commercially interesting trait in a plant by genetic manipulation can be a lengthy procedure dependent on different factors. The actual transformation and regeneration of genetically transformed plants are only the first in a series of selection steps, which include extensive genetic characterization, breeding, and evaluation in field trials, eventually leading to the selection of an elite event.
The unequivocal identification of an elite event is becoming increasingly important in view of discussions on Novel Food/Feed, segregation of GMO and non-GMO products and the identification of proprietary material. Ideally, such identification method is both quick and simple, without the need for an extensive laboratory set-up. Furthermore, the method should provide results that allow unequivocal determination of the elite event without expert interpretation, but which hold up under expert scrutiny if necessary.
GAT-ZM1 was selected as an elite event in the development of corn resistant to the herbicide Liberty®, by transformation of corn with plasmid pUC/Ac comprising the pat gene encoding tolerance to phosphinothricin. It is commercially sold as. Liberty Link® maize, such as, for instance, Liberty Link® A6460LL sold by AgriGold/Akin Seed Company. The tools for use in simple and unequivocal methods for identification elite event GAT-ZM1 in biological samples are described herein.
SUMMARY OF THE INVENTION
The present invention relates to methods for identifying elite event GAT-ZM1 in biological samples, which methods are based on primers or probes which specifically recognize the 5′ and/or 3′ flanking sequence of GAT-ZM 1.
More specifically, the invention relates to a method comprising of amplifying a sequence of a nucleic acid present in biological samples, using a polymerase chain reaction with at least two primers, one of which recognizes the 5′ or 3′ flanking region of GAT-ZM1, the other which recognizes a sequence within the foreign DNA, to obtain a DNA fragment of between 100 and 350 bp. Preferably, the primers recognize a sequence within the 5′ flanking region of GAT-ZM1, most preferably within the 5′ flanking region of SEQ ID No. 6, and a sequence within the foreign DNA, respectively. Especially preferably, the primer recognizing the 5′ flanking region comprises the nucleotide sequence of SEQ ID No 11 and the primer recognizing a sequence within the foreign DNA comprises the nucleotide sequence of SEQ ID No 12 described herein.
The present invention more specifically relates to a method for identifying elite event GAT-ZM1 in biological samples, which method comprises amplifying a sequence of a nucleic acid present in a biological sample, using a polymerase chain reaction with two primers having the nucleotide sequence of SEQ ID No 11 and SEQ ID No 12 respectively, to obtain a DNA fragment of between 180 and 220 bp, preferably of about 200 bp.
The present invention further relates to the specific flanking sequences of GAT-ZM1 described herein, which can be used to develop specific identification methods for GAT-ZM1 in biological samples. More particularly, the invention relates to the 5′ and or 3′ flanking regions of GAT-ZM1 which can be used for the development of specific primers and probes. The invention further relates to identification methods for the presence of GAT-ZM1 in biological samples based on the use of such specific primers or probes.
The invention further relates to kits for identifying elite event GAT-ZM1 in biological samples, said kits comprising at least one primer or probe which specifically recognizes the 5′ or 3′ flanking region of GAT-ZM1.
Preferably the kit of the invention comprises, in addition to a primer which specifically recognizes the 5′ or 3′ flanking region of GAT-ZM1, a second primer which specifically recognizes a sequence within the foreign DNA of GAT-ZM1, for use in a PCR identification protocol. Preferably, the kit of the invention comprises two specific primers, one of which recognizes a sequence within the 5′ flanking region of GAT-ZM1, most preferably within the 5′ flanking region of SEQ ID No. 6, and the other which recognizes a sequence within the foreign DNA. Especially preferably, the primer recognizing the 5′ flanking region comprises the nucleotide sequence of SEQ ID No 11 and the primer recognizing the transgene comprises the nucleotide sequence of SEQ ID No 12 described herein.
The invention firther relates to a kit for identifying elite event GAT-ZM1 in biological samples, said kit comprising the PCR primers having the nucleotide sequence of SEQ ID No. 11 and SEQ ID No. 12 for use in the GAT-ZM1 PCR identification protocol described herein.
The invention also relates to a kit for identifying elite event GAT-ZM1 in biological samples, which kit comprises a specific probe having a sequence which corresponds (or is complementary to) a sequence having between 80% and 100% sequence identity with a specific region of GAT-ZM1 . Preferably the sequence of the probe corresponds to a specific region comprising part of the 5′ or 3′ flanking region of GAT-ZM1. Most preferably the specific probe has (or is complementary to) a sequence having between 80% and 100% sequence identity to the sequence between nucleotide 286 and 466 of SEQ ID No. 6.
The methods and kits encompassed by the present invention can be used for different purposes such as, but not limited to the following: to identify GAT-ZM1 in plants, plant material or in products such as, but not limited to food or feed products (fresh or processed) comprising or derived from plant material; additionally or alternatively, the methods and kits of the present invention can be used to identify transgenic plant material for purposes of segregation between transgenic and non-transgenic material; additionally or alternatively, the methods and kits of the present invention can be used to determine the quality (i.e. percentage pure material) of plant material comprising GAT-ZM1.
The invention further relates to the 5′ and/or 3′ flanking regions of GAT-ZM1 as well as to the specific primers and probes developed from the 5′ and/or 3′ flanking sequences of GAT-ZM1.
DETAILED DESCRIPTION
The incorporation of a recombinant DNA molecule in the plant genome typically results from transformation of a cell or tissue (or from another genetic manipulation). The particular site of incorporation is either due to “random” integration or is at a predetermined location (if a process of targeted integration is used).
The DNA introduced into the plant genome as a result of transformation of a plant cell or tissue with a recombinant DNA or “transforming DNA” is hereinafter referred to as “foreign DNA” comprising one or more “transgenes”. Thus, foreign DNA may comprise both recombinant DNA as well as newly introduced, rearranged DNA of the plant. However, the term “plant DNA” in the context of the present invention will refer to DNA of the plant which is found in the same genetic locus in the corresponding wild-type plant. The foreign DNA can be characterized by the location and the configuration at the site of incorporation of the recombinant DNA molecule in the plant genome. The site in the plant genome where a recombinant DNA has been inserted is also referred to as the “insertion site” or “target site”. Insertion of the recombinant DNA into the plant genome can be associated with a deletion of plant DNA, referred to as “target site deletion”. A “flanking region” or “flanking sequence” as used herein refers to a sequence of at least 20 bp, preferably at least 50 bp, and up to
Aventis CropScience N.V.
Jones W. Gary
Souaya Jehanne
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