Use of selective DNA fragment amplification products for hybridization-based genetic fingerprinting, marker assisted selection, and high-throughput screening
AssigneePioneer Hi-Bred International, Inc.
InventorsRhonda J. McCasky Feazel, Timothy G. Helentjaris, Sharon E. Malmberg, Barry A. Martin
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Abstract
Methods of genotyping amplified mixtures of DNAs, nucleic acid markers and methods of obtaining markers, kits, recombinant plants, positional cloning and integrated systems for making genotypes and assessing hybridizations are provided. These features are applicable to DNA fingerprinting, marker assisted selection, genotyping, cladistic analysis of variance, and high throughput laboratory screening methods.
Claims20
A composition comprising at least two marker nucleic acids, which specifically hybridize to a nucleotide polymorphism and uniquely maps to a single site in a haploid genomic DNA of a plant or animal, and an amplified mixture of DNA isolated from a biological source, wherein the amplified mixture comprises a heterogeneous amplified mixture comprising a plurality of target nucleic acids comprising polymorphisms in amplified form and wherein the amplified mixture of DNA is made by cleaving a biological genomic DNA sample with at least one restriction enzyme, thereby providing DNA restriction fragments;
- ligating an adaptor nucleic acid to the DNA restriction fragments;
- hybridizing primers comprising a subsequence complementary to the adaptor nucleic acid;
- and,
- extending the primers with a thermostable DNA polymerase, thereby providing the amplified mixture of DNA comprising a selectively amplified mixture of DNAs.
A method of characterizing a nucleic acid, comprising:
- providing at least one probe which hybridizes to a marker in linkage disequilibrium with a polymorphism;
- amplifying a mixture of nucleic acids comprising a plurality of target nucleic acids comprising polymorphisms, thereby providing a heterogeneous amplified nucleic acid mixture comprising the plurality of target nucleic acids in amplified form;
- and,
- hybridizing the at least one probe to the amplified nucleic acid mixture, thereby detecting at least one target nucleic acid in amplified form.
A method of mapping a polymorphic genetic marker, comprising the steps of:
- (i) providing a mixture of nucleic acids from biological samples;
- (ii) amplifying the mixture of nucleic acids, thereby providing an amplified mixture comprising a plurality of polymorphic nucleic acids in amplified form;
- (iii) identifying a set of differentially amplified nucleic acids in the amplified mixture;
- and,
- (iv) mapping at least one of the differentially amplified nucleic acids to a unique genetic polymorphism, thereby providing a marker for the polymorphism.
A high-throughput method of selecting polymorphic variants by marker assisted selection, the method comprising the steps of:
- (i) providing a mixture of nucleic acids from a biological source, wherein the mixture of nucleic acids is a heterogeneous amplified mixture comprising a plurality of target nucleic acids comprising polymorphis in amplified form, the mixture optionally comprising a first target nucleic acid which hybridizes to a first marker nucleic acid which hybridizes to a first locus comprising a first nucleotide polymorphism;
- and,
- (ii) selecting the biological source for the presence or absence of the fist target nucleic acid in the mixture of nucleic acids, said presence or absence measured by hybridization of the marker nucleic acid to the mixture, thereby selecting for the presence or absence of the first nucleotide polymorphism.
Dependencies are inferred from the claim text.