Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-11-30
2004-05-11
Whisenant, Ethan (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300
Reexamination Certificate
active
06733974
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of plant molecular biology, more specifically the invention relates to composition and assays for detecting the presence of specific genetic constructs in plant transformation events in a DNA sample from a cotton plant or other plant species.
BACKGROUND
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. Pat. No. 5,004,863. One such introduced DNA sequence important in cotton production is the cauliflower mosaic virus 35S promoter (CaMV35 or 35S) sequence. The promoter from the cauliflower mosaic virus is one of the most abundantly used genetic elements in genetically transformed crops. An important gene is the neomycin phosphotransferase sequence (nptII). The nptII sequence was originally isolated from the bacterial transposon 5. The nptII sequence has been used in many genetically transformed crops as a marker sequence. These DNA sequences have been introduced into cotton plants and are found in commercially available transformed varieties of cotton that are under production.
The expression of foreign gene sequences 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., Ann. Rev. Genet 22:421-477, 1988). For a number of reasons, it is often desirable to screen for specific DNA sequences in order to confirm the presence of an event of interest. For example, it has been observed in plants and in other organisms that there may be a wide variation in levels of expression of an introduced genes among 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 DNA construct(s). There is also an increasing interest in sampling lots of plant-based material for identity preservation, that is, to detect whether the material being tested contains any, all or no genetically-transformed plant material and/or the source of any transformed plant material identified in a given lot that is being tested or sampled. For this and other reasons, it is often desirable to screen or assay for a specific genetic transformation event.
One example of when it would be advantageous to be able to detect the presence of a particular event is 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 various regulatory requirements, for example regulations requiring the premarket approval or labeling of foods derived from recombinant crop plants.
It is possible to detect the presence of a genetic transformation event by any well known nucleic acid detection method including DNA amplification methods, such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc. As a result, such methods may not be useful for discriminating between different genetic transformation events that possess more than one element, particularly those produced using the same DNA construct unless additional information is known. An event-specific 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 by PCR using a primer set spanning the junction between the insert and flanking DNA, specifically one primer that included sequence from the insert and a second primer that included sequence from flanking DNA.
However, these currently available methods of detecting genetic transformation events in plant material involve the use of assays that are not specific or robust enough to differentiate among specific genetic transformation events. Commonly such previously available methods involve performing multiple detection assays, each assay specific for just one genetic construct or sequence, in order to detect the presence of transgenic DNA. For example, one assay for detection of the 35S promoter that is currently in use for detection of transgenic plant material is designed to detect the presence of the 35S promoter in a sample. The problem with this is that a sample can be positive for the 35S promoter in a detection assay if it contains any one of the commercial cotton genetic transformation events, or if the sample contains another of the at least twenty-two products that currently use the 35S promoter sequence, or if the sample has been contaminated with a virus that has a similar sequence to the 35S promoter (a false positive result). Such a 35S promoter detection assay is considered to be not specific enough to say with certainty the source identity of a positive result. An assay for the 35S promoter alone cannot definitively determine whether a sample that gives a positive result does contain, for example, a sample from a transformed cotton plant or represents some other transgenic sample or represents a plant infected by a virus with a similar sequence. To detect even the current commercial cotton genetic transformation events, at least three separate event-specific PCR assays would need to be performed. More specific PCR assays (with lower false positive levels) to detect the current commercial cotton transgenic events are still needed.
SUMMARY
A single assay to detect the presence of specific genetic transformation events in a sample lot of plant material such as cotton seed, the primers for such an assay and methods of using the assay are provided. DNA sequences are provided that comprise unique and advantageous primers that produce unique and unexpected target amplicons that span a junction sequence of an event comprising the 35S promoter and nptII coding sequences; the primers are identified as SEQ ID NO:1 and SEQ ID NO:2. Sequences highly homologous to these primers are also useful in the assay, as are the complements to each, as one would expect.
The invention accomplishes the designing of a single generic assay that detects all three of the current commercial cotton transgenic events and is more specific than current promoter or gene-specific assays. The assay methods also detect the presence of a DNA construct comprising the 35S promoter and nptII sequence in close proximity and in the same orientation in other species of plants.
The assay of the present invention uses one primer anchored in the 35S promoter and one primer anchored in the nptII sequence. The resulting amplicon of these primers span the junction region between the 35S promoter and the nptII sequence in the constructs. Different constructs of these elements will produce different amplicons. The constructs used in making the current cotton products were different and the present assay can differentiate between the two constructs used.
It is highly unlikely that any potential contaminants in a seed or other plant-material-containing sample other than from a transgenic source could produce a positive result using this assay since the 35S promoter sequence and nptII sequence are not found together in non-transgenic organisms. It was surprisingly discovered that although many primers can be generated to anchor in each element of the sequence, such primers usually are not specific and robust in their performance and produce both false positive results and/or false negative results to when used in an assay.
The primers of the present invention overcome this problem. The amplicons produced using these primers are specifically and robustly diagnostic for the nptII/35S construct used to m
Bonner Grace L.
Lawson E. Clifford
Monsanto Technology LLC
Whisenant Ethan
LandOfFree
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