Use of selective DNA fragment amplification products for hybridization-based genetic fingerprinting, marker assisted selection, and high-throughput screening

• US 6,100,030 A
• Filed: 01/09/1998
• Issued: 08/08/2000
• Est. Priority Date: 01/10/1997
• Status: Expired due to Term

• Current Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Original Assignee: Pioneer Hi-Bred International (Corteva, Inc.)
• Inventors: Helentjaris, Timothy G., Malmberg, Sharon E., Martin, Barry A., McCasky Feazel, Rhonda J.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US09/005,205
• Time in Patent Office: 942 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2600/13 Plant traits
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

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.

Claims

1. A method of characterizing a biological nucleic acid, comprising:

providing at least one probe which hybridizes to a marker in linkage disequilibrium with a polymorphism;

amplifying a mixture of biological DNA comprising a plurality of target nucleic acids comprising polymorphisms, thereby providing a heterogeneous amplified DNA mixture comprising the plurality of target nucleic acids in amplified form; and

,

hybridizing the at least one probe to the amplified DNA mixture, thereby detecting at least one target nucleic acid in amplified form.

2. The method of claim 1, wherein the mixture of biological DNA is amplified using an amplification technique selected from the group consisting of cloning, PCR, LCR, TAS, 3SR, NASBA and Qβ

amplification.

3. The method of claim 1, wherein the mixture of biological DNA is selected from the group of DNAs consisting of genomic DNA, and cDNA.

4. The method of claim 1, wherein the amplified DNA mixture is made by cleaving the mixture of biological DNA 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 a selectively amplified mixture of DNAs.

5. The method of claim 3, wherein the primer comprises an arbitrary 3'"'"' nucleotide which is not complementary to the adaptor nucleic acid.

6. The method of claim 3, wherein the primer comprises an arbitrary 3'"'"' nucleotide and the method further comprises further amplifying the selectively amplified mixture of DNAs using an amplification primer comprising 3 arbitrary nucleotides at the 3'"'"' end of the primer, which 3 arbitrary nucleotides are not complementary to the adaptor nucleic acid.

7. The method of claim 1, wherein the probe is an oligonucleotide between about 8 and about 100 nucleotides in length, which probe specifically hybridizes to the polymorphism.

8. The method of claim 1, wherein the probe is fixed to a solid support and the amplified DNA mixture is hybridized to the probe.

9. The method of claim 8, wherein the probe is a member of an array of probes, which array comprises additional probes which hybridize to one or more markers in linkage disequilibrium with at least one polymorphism.

10. The method of claim 8, wherein the solid support is selected from the group consisting of a nylon membrane, a nitrocellulose membrane, a plastic surface, a glass surface, and a microtiter dish.

11. The method of claim 1, wherein the amplified DNA mixture is fixed to a solid support and the probe is hybridized to the amplified DNA mixture.

12. The method of claim 1, wherein the amplified DNA mixture is fixed to a solid support in an array of fixed amplified DNA mixtures and the probe is hybridized to the array in a dot blot format.

13. The method of claim 1, wherein the amplified DNA mixture is heterogeneous by size.

14. The method of claim 1, wherein the amplified DNA mixture is homogeneous by size and heterogeneous by sequence.

48. The method of claim 1, wherein a plurality of probes is hybridized to the amplified DNA mixture.

15. A method of mapping a polymorphic genetic marker, comprising the steps of:

(i) providing a mixture of restriction enzyme-digested nucleic acids from biological samples;

(ii) amplifying the mixture of restriction enzyme-digested nucleic acids;

(iii) identifying a set of differentially amplified nucleic acids in the 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.

16. The method of claim 15, further comprising hybridizing a probe nucleic acid, which probe nucleic acid hybridizes under stringent conditions to a target nucleic acid comprising the polymorphism, to a mixture of DNA amplified from a biological source of DNA comprising the polymorphism, thereby identifying the polymorphism in the biological source of DNA.

17. The method of claim 16, wherein the biological source of DNA is selected from the group consisting of cDNA, genomic DNA isolated from a plant, genomic DNA isolated from a plant extract, genomic DNA isolated from an isolated plant tissue, genomic DNA isolated from an isolated plant tissue extract, genomic DNA isolated from a plant cell culture, genomic DNA isolated from a plant cell culture extract, genomic DNA isolated from a recombinant cell comprising a nucleic acid derived from a plant, genomic DNA isolated from a plant seed, genomic DNA isolated from an extract of a recombinant plant cell comprising a nucleic acid derived from a plant, genomic DNA isolated from an animal, genomic DNA isolated from an animal extract, genomic DNA isolated from an isolated animal tissue, genomic DNA isolated from an isolated animal tissue extract, genomic DNA isolated from an animal cell culture, genomic DNA isolated from an animal cell culture extract, genomic DNA isolated from a recombinant animal cell comprising a nucleic acid derived from an animal, genomic DNA isolated from an animal egg, genomic DNA isolated from an extract of a recombinant animal cell, DNA isolated from a mitochondria, and DNA isolated from a chloroplast.

18. The method of claim 15, wherein the set of differentially amplified nucleic acids comprises about 10 or more differentially expressed nucleic acids.

19. The method of claim 15, wherein more than one of the differentially amplified nucleic acids are mapped, thereby providing a set of markers of unique genetic polymorphisms.

20. The method of claim 19, wherein at least one of the differentially amplified nucleic acids maps proximal to a QTL.

21. The method of claim 20, wherein the QTL is selected from the group consisting of yield, grain moisture, grain oil, root lodging, stalk lodging, plant height, ear height, disease resistance, insect resistance, resistance to soybean cyst nematode, resistance to brown stem rot, and resistance to phytopthora rot.

22. The method of claim 19, further comprising marker assisted selection with the set of markers.

23. The method of claim 19, further comprising marker assisted selection with the set of markers, wherein at least one of the differentially amplified nucleic acids maps proximal to a QTL and wherein the method further comprises backcrossing a plant comprising the QTL to a selected plant to introgress the QTL into progeny of the selected plant.

24. The method of claim 19, further comprising marker assisted selection with the set of markers, wherein the set of markers is hybridized to an array of amplified mixtures of DNA from a biological source.

25. The method of claim 19, further comprising marker assisted selection with the set of markers, wherein the set of markers is provided in an array and a mixture of DNA from a biological source is hybridized to the array.

26. The method of claim 19, further comprising genotyping the biological source of DNAs with the set of markers.

27. The method of claim 22, wherein the set of marker nucleic acids is provided in kit form, said kit optionally comprising one or more component selected from the components consisting of a container, instructional materials, one or more control nucleic acids complementary to the markers, and recombinant cells comprising one or more target nucleic acids.

28. The method of claim 22, wherein the selection is performed on a plant selected from the group consisting of corn, canola, soybean, wheat, sunflower, and sorghum.

29. The method of claim 15, further comprising positional cloning of a selected nucleic acid in linkage disequilibrium with the nucleotide polymorphism.

30. The method of claim 29, further comprising transformation of a plant with the cloned selected nucleic acid, thereby providing a transgenic plant.

31. The plant produced by the method of claim 30.

32. 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 amplified from a biological source, the mixture optionally comprising an amplified 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 first target nucleic acid in the mixture of amplified nucleic acids, said presence or absence measured by hybridization of the marker nucleic acid to the amplified mixture, thereby selecting for the presence or absence of the first nucleotide polymorphism.

33. The method of claim 32, wherein the biological source is selected from the group consisting of a plant, a plant extract, an isolated plant tissue, an isolated plant tissue extract, a plant cell culture, a plant cell culture extract, a recombinant cell comprising a nucleic acid derived from a plant, a plant seed, an extract of a recombinant plant cell comprising a nucleic acid derived from a plant, an animal, a animal extract, an isolated animal tissue, an isolated animal tissue extract, an animal cell culture, an animal cell culture extract, a recombinant animal cell comprising a nucleic acid derived from an animal, an animal egg, an extract of a recombinant animal cell, a DNA isolated from a mitochondria and a DNA isolated from a chloroplast.

34. The method of claim 32, wherein the amplified mixture of nucleic acids is provided by a technique selected from the group consisting of:

PCR, LCR, TAS, 3SR NASBA, Qβ

amplification and cloning of the target nucleic acid.

35. The method of claim 32, the mixture of amplified DNAs optionally further comprising a second amplified target nucleic acid which hybridizes to a second marker nucleic acid which hybridizes to a second locus comprising a second nucleotide polymorphism, the method further comprising selecting the biological source for the presence or absence of the second target nucleic acid in the mixture of amplified DNAs, thereby selecting for the presence or absence of the second nucleotide polymorphism.

36. The method of claim 35, wherein the first and second marker nucleic acids are probe nucleic acids in an array of nucleic acid polymers fixed to a solid support.

37. The method of claim 32, the method further comprising selecting a second biological source for the presence or absence of the first nucleotide polymorphism.

38. The method of claim 37, wherein the first and second biological sources are selected for the presence of the first nucleotide polymorphism in parallel.

39. The method of claim 37 further comprising parallel selection of n additional biological sources for the presence or absence of the first nucleotide polymorphism, where n is an integer equal to or larger than 1.

40. The method of claim 39, wherein amplified mixtures of DNAs from the additional biological sources are individually fixed to one or more solid supports and labeled marker nucleic acids are hybridized to the fixed mixtures of amplified DNAs.

41. The method of claim 39, wherein marker DNAs are fixed to one or more solid support, the amplified mixtures of DNAs are labeled, and the amplified mixtures of DNAs are hybridized to the solid support.

42. The method of claim 37 further comprising parallel selection of n additional biological sources for the presence or absence of the first nucleotide polymorphism, where n is an integer between about 1 and about 4,000.

43. The method of claim 32, wherein the first polymorphic nucleotide is in linkage disequilibrium with a QTL.

44. The method of claim 32, wherein the first polymorphic nucleotide is in linkage disequilibrium with a QTL and wherein the method further comprises backcrossing a plant or animal comprising the polymorphic nucleotide to a selected plant or animal to introgress the QTL into the selected plant or animal.

45. The method of claim 32, wherein the presence or absence of the target nucleic acid is determined by hybridizing the probe to the mixture using a hybridization technique selected from the group consisting of Southern blotting, northern blotting, and array-dependent nucleic acid hybridization on a nucleic acid polymer array.

46. The method of claim 32, the method further comprising detection of a plurality of amplified target nucleic acids in the amplification mixture, which target nucleic acids are genetically linked to a plurality of polymorphic nucleotides at a plurality of unique loci.

47. The method of claim 46, wherein the plurality of amplified targets are detected using an array of selection probes, which probes comprise polymorphic nucleotides.