Data processing: measuring – calibrating – or testing – Measurement system in a specific environment – Biological or biochemical
Reexamination Certificate
1999-04-02
2002-11-19
Brusca, John S. (Department: 1631)
Data processing: measuring, calibrating, or testing
Measurement system in a specific environment
Biological or biochemical
C435S006120, C536S023100
Reexamination Certificate
active
06484105
ABSTRACT:
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
Not applicable.
BACKGROUND OF THE INVENTION
In recent years, genomic sequencing of several organisms has been completed and genomic sequencing of many other organisms has also been initiated. Furthermore, the complete genomic sequences of many organisms, including human, some model organisms such as
C. elegans,
Drosophila and Arabidopsis, as well as some economically important crops and animals will be known within the next decade. Many of the genes identified from this research, however, will have unknown functions.
One approach to study gene functions is to knock out a gene of an organism and find out what happens to the mutant organism. Studying the mutant organism often leads to better understanding of the functions of the gene. Also, gene knockouts allow for the specifically elimination or change of undesirable traits.
Currently there are technologies available to create knock-out animals through homologous recombination. See, e.g., Galli-Taliadoros, et al.
J. Immunol. Methods
181(1):1-15 (1995); Buerstedde, et al.,
Cell,
67:179-88 (1991); Jasin, et al.,
Genes and Devel.,
2:1353-63 (1988); Sedivy, et al.,
Proc. Natl. Acad. Sci. USA,
86:227-31 (1989). Such technologies are expensive and inefficient. For unknown reasons, however, the frequency of homologous recombination in plants is so low that it is not possible to create a knock-out plant through homologous recombination. Although some researchers have succeeded in lowering expression levels of certain plant genes by making transgenic plants expressing antisense RNA of those genes, they can not completely eliminate the expression and function of those genes. In many cases, complete elimination of the function of a gene is needed. Also, it is currently either very difficult or impossible to make transgenic plants in many crop species.
Bensen et al.
Plant Cell
7(1): 75-84 (1995) teach a method of identifying transposon insertion mutants in known maize gene sequences by amplifying the region between a transposon sequence and a particular gene sequence. This method, however, is limited to the few plant systems with characterized transposon systems or requires the introduction of such systems in heterologous plants. This method therefore does not provide a universal method for identification of mutations in known sequences of any plant species.
Therefore a need exists for obtaining and detecting a genetic lesion in a known plant gene sequence. Ideally, this method should be useful in as broad a range of plants as possible and therefore should not depend on such criteria as availability of transformation methods.
SUMMARY OF THE INVENTION
The present invention provides a method for identifying a plant containing a lesion in a gene sequence. The method comprises providing a collection or pool of nucleic acid samples wherein each sample is collected from an individual plant. Typically the individual plants used in the method are derived from a mutagenized population of source plants, thereby insuring genetic diversity within the population.
The method of the invention also provides a database that associates each nucleic acid sample with the source plant from which the nucleic acid sample was derived.
In addition, the method of the invention includes amplifying the collection of nucleic acids using primers that specifically hybridize to known polynucleotide sequences that flank the gene sequence in the wild type chromosome. In one embodiment, the amplification includes an extension step that is shortened to selectively amplify a nucleic acid with the lesion. For instance, the extension time can be between 0.5 and two minutes at 72° C. In one embodiment, the extension time is 1.5 minutes.
After the nucleic acids are amplified, a genetic lesion within the gene sequence of one of the samples is detected. For instance, the lesion can be detected as a change in size of the amplification product. Once the nucleic acid sample containing the lesion is identified, the database is used to identify the source plant from which the nucleic acid sample was derived.
In one embodiment, the lesion is a deletion.
In another embodiment, the collection of nucleic acids is divided into subset pools before the lesion is detected. For example, the pools can contain nucleic acids from about 100 different to about 3000 different source plants. In another embodiment, the pools can contain at least about 1500 different source plants.
The plurality of source plants or the progenitors of the source plants can, for example, be contacted with a mutagen. In one embodiment, the mutagen is fast neutrons.
In one embodiment, the distance between the known nucleotide sequences in the wild type plant is between about three kilobases and seven kilobases.
The method of the invention can be performed, for instance, on a plurality of source plants such as rice, tomato or Arabidopsis. In one embodiment, the number of source plants is about 10,000 to about 50,000.
Included in the invention is a plant identified by the method described above.
In a preferred embodiment of the invention, a source plant carrying a deletion in a gene sequence is identified. In this embodiment, a collection of nucleic acids from a plurality of mutagenized plants, at least one of which carries a deletion in the gene sequence, is provided. A database is also provided that associates each nucleic acid in the collection with the nucleic acid's source plant. The collection of nucleic acids is then amplified. The amplification comprises providing primers that specifically hybridize to polynucleotide sequences from about 3 kb to about 7 kb apart in the wild type chromosome and that specifically hybridize to polynucleotide sequences within or flanking the gene sequences. The amplification further comprises an extension step of between about 0.5 minutes to about two minutes at 72° C., thereby preferentially amplifying the nucleic acids with the deletion. The presence of the nucleic acid with the deletion is confirmed by comparing the amplification product with an amplification product from a wild type plant. The database is then used to identify the source plant with the deletion, thereby providing a plant with a deletion in the gene sequence for evaluation of a mutant phenotype associated with the deletion.
Definitions
A “gene sequence” refers to a nucleic acid sequence that comprises a sequence or subsequence from an open reading frame and/or regulatory sequences in genomic DNA or a cDNA.
A “wild type chromosome” refers to a polynucleotide sequence that exists in a plant before human-initiated mutagenesis is performed. “Human initiated mutagenesis” does not include, for instance, selection of agronomically valuable traits through selective breeding, but does include the introduction of mutagenic compounds or elements to promote genetic diversity in a population. Such mutagenic compounds include, e.g., transposons.
A “source plant” is a particular plant from which nucleic acids were harvested or the plant's genetic equivalent. For the purposes of this invention, genetically equivalent plants are those plants from which the same specific genetic lesion can be recovered. Genetically equivalent plants includes siblings or progeny from a plant of interest. Thus, if M1 seeds are mutagenized, grown into M1 plants and their M2 seed are harvested, pooled M2 seeds from the same parent are the same “source plant” because a specific mutation of one M2 plant that was received from an M1 parent is likely to be found in a pool of M2 siblings from the same M1 parent. Similarly, if an M2 plant has a particular genetic lesion, pools of M3 progeny from the M2 plant will also likely carry that lesion.
A “lesion” can be any molecular alteration of a nucleic acid relative to wild type plant nucleic acids. For instance, a lesion can be a deletion, inversion, insertion, duplication or a rearrangement in a nucleic acid sequence. The lesion is preferably deletion.
“Progenitors of the plurality of source plants” ar
Holman Christopher M.
Kruse Norman J.
Maxyag, Inc.
Moran Marjorie A.
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