Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-07-20
2003-05-27
Siew, Jeffrey (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120, C435S091100, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330
Reexamination Certificate
active
06569648
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, it concerns methods and compositions for the identification and selection of insertional mutants.
2. Description of Related Art
Mutants are powerful tools in the investigation of physiological, developmental, and cell biological processes. Starting with a phenotypic mutant generated by chemical mutagenesis, it is possible to use a genetic map-based strategy to clone a gene (Arondel et al., 1992). Mutations derived from insertional mutagenesis are particularly useful in that they provide “tagged” copies of the mutated gene which may readily be cloned (Yanofsky et al., 1990). However, molecular genetic techniques have advanced such that today most genes are cloned and sequenced long before their function is characterized genetically (Newman et al., 1994). For many genes, phenotypic screens are not available, and mutations which cause lethality remain undetectable. What has been missing is a simple and reliable strategy to go from a gene or protein sequence to the identification of specific mutants.
One solution to problems associated with mutant identification was to use the polymerase chain reaction (PCR) to screen for P-element mutations in sequenced genes of Drosophila (Ballinger et al., 1989; Kaiser et al., 1990). This approach also enhanced the genetics of Caenorhabditis (Rushforth et al., 1993; Zwaal et al., 1993), where transposable element mutations are now commonly isolated for known gene sequences. In these systems, transposon-induced mutations are isolated for known gene sequences by the general strategy known as “site-selected” mutagenesis. Basically, the method relies on the power of PCR to amplify a collection of specific junction fragments between an inserted element and a known target gene sequence from large pools of randomly inserted elements. One primer is used which is homologous to the end of the inserted element with its 3′ end facing outward and one primer within the target gene is used to amplify the sequences at the junction of the insertion. In plants, similar approaches have been used to identify insertion mutations in Petunia, using the transposon dTph1 (Koes et al. 1995), and in Arabidopsis using collections of T-DNA transformed lines (Krysan et al., 1996; Mckinney et al., 1995). In Krysan et al. (1996), 9100 independent T-DNA-transformed Arabidopsis lines (averaging 1.4 insertions per genome) were subjected to site-selected mutagenesis and 17 T-DNA insertions within 63 genes were identified.
While techniques based on the gene-specific amplification of insertional junctions have been useful in the isolation of a number of mutants, they have had limited success in applications toward large-scale genomic investigations. The need for individual amplifications of each gene being investigated represents a significant hindrance when seeking to identify more than a small number of insertional mutants. There is, therefore, a great need in the art for a method by which large numbers of insertional mutants may be rapidly and efficiently identified.
SUMMARY OF THE INVENTION
The present invention seeks to overcome deficiencies in the prior art by providing a highly efficient method for selecting insertion events. Therefore, one apsect of the current invention is a method for identifying an insertion event in a genome comprising the steps of: (a) preparing a first DNA composition enhanced for a plurality of insertion junctions; (b) preparing at least a first detectable array including the first DNA composition; and (c) detecting the insertion event from the first array. The step of preparing a first DNA composition may comprise amplification of insertion junctions with inverse PCR, vectorette PCR, primer-adapted PCR, AIMS or any other suitable procedure. The method can further comprise preparing at least a second DNA composition, and additionally any greater number of DNA compositions desired by the user of the invention. The additional DNA compositions may be prepared on the same, or other arrays, as desired by the user of the invention.
In another aspect of the invention, the detectable array can comprise the first and second DNA compositions arranged on a solid support. The solid support can be a microscope slide, and the insertion event can be detected by hybridization with a fluorescently labeled probe comprising cloned DNA, and/or be detected by hybridization with a probe labeled with an antigen, where the antigen is detected with a molecule which binds the antigen. Alternatively, the insertion event can be detected by PCR. In another embodiment of the invention, the array has a solid support comprising a nitrocellulose filter, and the insertion event can be detected by hybridization with a radioactively-labeled probe comprising cloned DNA. The method of detecting can further comprise hybridization of a gene-specific probe to the array. In particular embodiments, the DNA compositions of the array will comprise DNA which has been pooled from multiple individuals. The DNA in the compositions can be derived from potentially any species, including DNA from plants, animals, prokaryotes and lower eukaryotes. In particular embodiments, the DNA may be from a monocot plant, and may further defined as from maize, rice, wheat, barley, sorghum, oat, or sugarcane. In other embodiments, the monocot DNA is maize DNA. The plant DNA may also be dicot DNA, and may be derived from a species selected from the group consisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. In particular embodiments of the invention the dicot plant DNA is
Arabidopsis thaliana
DNA. In still other embodiments the DNA is animal DNA.
Still yet another aspect of the invention provides a method of determining the function of a DNA sequence. In particular embodiments of the invention the method comprises the steps of: (a) amplifying a plurality of insertion junctions from a DNA composition comprising insertion mutations; (b) creating at least a first array comprising said insertion junctions; (c) detecting at least a first mutation in said DNA sequence from said array using a primer or probe specific to said DNA sequence; and (d) determining the function of said DNA sequence by comparing individuals comprising said mutation in said DNA sequence to corresponding individuals lacking said mutation in said DNA sequence. In the method, the DNA composition may comprise plant DNA. In particular embodiments the plant DNA may be further defined as monocot plant DNA, and may be still further defined as derived from a species selected from the group consisting of maize, rice, wheat, barley, sorghum, oat, and sugarcane. In particular embodiments, the monocot DNA comprises maize DNA. The plant DNA can also comprise dicot plant DNA, and may be still further defined as derived from a species selected from the group consisting of cotton, tobacco, tomato, soybean, sunflower, oil seed rape (canola), alfalfa, potato, strawberry, onion, broccoli, Arabidopsis, pepper, and citrus. In particular embodiments, the DNA composition is
Arabidopsis thaliana
DNA.
Still yet another aspect of the invention provides a method for isolating a plant comprising a desired integration event. In particular embodiments of the invention, the method comprises the steps of: (a) integratively transforming a plurality of plants; (b) obtaining DNA from said plants; (c) amplifying a plurality of transgene insertion junctions from said DNA; (d) preparing at least a first array comprising said amplified insertion junctions; and (e) detecting a desired integration event with a probe or primer corresponding a preselected genomic region. In particular embodiments, the plant may be further defined as a monocot plant, and may be still further defined as derived from a species selected from the group consisting of maize, rice, wheat, barley, sorghum, oat, and sugarcane. In other embodiments, the mon
Chomet Paul S.
Dellaporta Stephen L.
DeKalb Genetics Corporation
Fulbright & Jaworski L.L.P.
Siew Jeffrey
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