Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-20
2002-03-19
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06358690
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
Not applicable.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
To be determined.
BACKGROUND OF THE INVENTION
One of the objectives of much genetic research is to identify the genes responsible for selected phenotypic traits in individuals. While much effort is being undertaken to develop genomic information from a large number of organisms, often the information about the function of a gene is more important than information as to the sequence of the gene itself. One way in which the function of individual genes is studied is to look for mutated versions of the gene of interest. Sometimes the search for mutated versions of a gene and the study of the mutated genes is referred to as “reverse genetics.” If one finds a gene which is mutated so as to render the mutated gene inoperative, one can discern what phenotypic change has been made to the organism that renders it different from organisms not carrying the mutated version of the gene. Such studies can often lead to insightful information about the function of the gene which has the mutation.
Various strategies have been developed for using reverse genetics to study the functioning of genes in the model plant species
Arabidopsis thaliana.
For example, one laboratory at the University of Wisconsin has created a large population of Arabidopsis plant lines each of which has been transformed using the transferred-DNA (T-DNA) from the bacterium
Agrobacterium tumefaciens,
which has the native ability to transfer its T-DNA into the genome of a plant cell. Since the mechanism for DNA transfer operated by Agrobacterium results in a random insertion of the transferred DNA into the plant genome, the use of the Agrobacterium transformation technique results in large populations of plants each of which has a potentially disruptive insert at a different place within the plant genome. The problem then becomes to identify the particular gene which has been disrupted within each of the plants and to determine what the function of the gene might have been prior to disruption.
Another technique for introducing mutations into genes in an individual species under study, as such as a plant, is to introduce transposable elements into the genome of the organism. Various transposable elements are known in plants and many are known to be immobilized in Arabidopsis plants. A transposable element transports itself within the genome to insert itself randomly within the genome of the plant. Such random insertion mutations also could help identify the function of genes by disrupting the function of those genes and permitting the disruption to be observed at a phenotypic level.
Another method used to produce random mutations in the genome of various species, such as plants, is to expose the tissues of the plant to ionizing radiation. Ionizing radiation is known to produce deletion mutations in the genome of many plant species. The deletions created range in size from less to 1 kilobase to large chromosomal scale deletions that remove entire arms of chromosomes. Inducing such deletions in the genome of a plant is as simple as exposing a bag of seeds to the ionizing radiation. The radiated seeds can then be planted and each resulting plant carries a different deletion mutation. By collecting the seeds of the resultant plants one can easily generate a large population of plants that carry deletion mutations throughout the entire genome. No special transformation or transposon protocols are needed, and one is limited only by the amount of space available to grow the plants.
Whichever method of mutation creation is used, the problem still becomes to rapidly localize the rare organisms that carry a mutant version of the particular gene of interest. Thus what is needed is a method for rapidly identifying relatively rare deletion mutations within a population of organisms, such as a plant.
BRIEF SUMMARY OF THE INVENTION
The present invention is summarized in that a technique based on a polymerase chain reaction (PCR) has been developed that permits the rapid and efficient characterization of deletion mutants in individual organisms.
The present invention is also summarized in a method that allows one to identify individual
Arabidopsis thaliana
plants which contain a single deletion mutation in their genome, the deletion resulting in a functional mutation of a gene of interest.
Other objects, advantages and features of the present invention will become apparent from the following specification.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
Not applicable.
DETAILED DESCRIPTION OF THE INVENTION
The method described here was developed for a situation in which one individual in a population of individuals of that species carries a deletion mutation in a particular gene, the function of which is sought to be studied. The sequence of the gene is known, and enough random deletion mutants have been made that the statistical likelihood is high that an individual exists in the population with a deletion mutation in the gene of interest, but the identity of that particular individual is not known. This method provides a very practical and efficient method that may be used to identify the individual with a deletion mutation in the desired locus.
The method is based on the polymerase chain reaction, or PCR, which is now a standard protocol of molecular biology and genetic laboratories. In a standard PCR reaction, the object is to amplify, or replicate exponentially copies of a particular sequence of a target DNA sequence possibly present in a sample. The PCR reaction in performed in a series of rounds, and in each round there are steps of denaturing, annealing and extending. In the denaturing step, the double stranded DNA is heated so as to denature the double stranded DNA so that it becomes single stranded. In the annealing step, the temperature is lowered so that conditions permit the annealing of specially made primers, which have sequences selected to bind to the target sequence, to the target DNA forming partially double stranded complexes. Then in the extension step, a DNA polymerase replicates the complement of the target strand by beginning at the double stranded complex of the primer and the target and extending the sequence of the primer using target sequence as a template. The product of each round of replication becomes a template for the next round. In the presence of excess primer, the target sequence is exponentially amplified. These three steps of denaturing, annealing, and extension are conducted repeatedly, each amplifying the complement of the target sequence, if it is present at all, until sufficient amplified DNA is made to be detectable by some detection procedure. In normal PCR, the object is to amplify the full length target sequence and thus the parameters of the process are normally arranged so as to maximize the yield of full length product made.
In the method of the present invention, the object is not to make full length target strands but to find, in essence, shorter sequences. The PCR process is adjusted by manipulating the three basic steps so that the shorter target sequences, representing the deletion mutations, are selectively amplified in preference over the full length sequences. This can be done most conveniently by limiting the time of the extension step in the process. Making this modification to standard PCR results in a process which selectively favors the amplification of shorter DNA species that still bind to the primers. In other words, this modified form of PCR reaction results in selective amplification of deletion mutant forms of native genes.
It is envisioned that this process will be used with pooled DNA from many individuals, for reasons of practical convenience. To screen large populations of individuals for presence of a desired deletion mutation, DNA from the individuals is obtained and processed in large pools, each pool containing the DNA from thousands of individuals. Then the pools can be tested for the presence of the deletion mutation in that pool.
Krysan Patrick John
Sussman Michael Richard
Horlick Kenneth R.
Quarles & Brady LLP
Strzelecka Teresa
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