Species-specific DNA-DNA hybridization probe prepared using chromosome size DNA
AssigneeThe United States of America as represented by the Secretary of Agriculture
InventorsTamas Torok, A. Douglas King, Jr., David R. Rockhold, Christina Royer
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Abstract
A species-specific DNA-DNA hybridization probe prepared using an isolated whole chromosome of a lower eukaryote as template. The probe is useful for the detection of conspecificity in lower eukaryotes selected from the group consisting of yeast and molds.
Claims3
A method for assaying a test sample for conspecificity in lower eukaryotes selected from the group consisting of yeasts and molds, comprising:
- (a) providing an isolate of double-stranded chromosomal DNA corresponding to substantially the entire DNA sequence of a chromosome in a yeast or mold;
- (b) denaturing said double-stranded chromosomal DNA to yield single-stranded DNA templates corresponding to substantially the entire DNA sequence of said chromosome;
- (c) random priming said single-stranded DNA templates with short chain primers in the presence of DNA polymerase and a nucleotide mixture to produce a probe which comprises a mixture of random-size copies of DNA molecules, said probe containing the DNA sequence information of substantially said entire chromosome;
- (d) contacting a test sample comprising a separated, denatured chromosome with said probe under hybridization conditions such that DNA sequences in the test sample which are substantially homologous with said probe are hybridized to produce a hybridization product;
- (e) separating said hybridization product from unhybridized material by washing under conditions of lower stringency;
- and
- (f) analyzing for said hybridization product.
A method for assaying a test sample for conspecificity in lower eukaryotes selected from the group consisting of yeasts and molds, comprising:
- (a) contacting a test sample comprising a separated, denatured chromosome a probe prepared by denaturing an isolated chromosomal DNA corresponding to substantially the entire DNA sequence of a chromosome in a yeast and mold to yield single stranded DNA templates and random priming said single-stranded DNA templates with short chain primers in the presence of DNA polymerase and nucleotide mixture to produce a probe which comprises a mixture of random-size copies of DNA molecules, said probe containing the DNA sequence information of substantially said entire chromosome and hybridizing under hybridization conditions such that DNA sequences in the test sample which are substantially homologous with said probe are hybridized to produce a hybridization product;
- (e) separating said hybridization product from unhybridized material by washing under conditions of low stringency;
- and
- (f) analyzing for said hybridization product.
Dependencies are inferred from the claim text.