Method and test kit for demonstrating genetic identity

• US 20030170705A1
• Filed: 01/27/2003
• Published: 09/11/2003
• Est. Priority Date: 01/30/2002
• Status: Abandoned Application

• Current Assignee: Boreal Plant Breeding, Ltd.
• Original Assignee: Boreal Plant Breeding, Ltd.
• Inventors: Paulin, Lars Goran, Schulman, Alan Howard
• Application Number:
  US10/351,934
• Publication Number:
  US 20030170705A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The present invention is related to a method and a test kit for demonstrating genetic identity, genetic diversity, genomic variations or polymorphisms, especially allelic variations, and also biodiversity within a defined population pool, with co-dominant scoring. The method and the test kit apply mobile elements (MEs) and are based on the use of one or more sets of optionally paired or parallel oligonucleotides, which are attached to a solid support. Each oligonucleotide sequence represents an insertion site junction of a mobile element (ME). The invention is also related to the use of the method and the test kit for phylogenetic studies, parenthood determinations, genotyping, haplotyping, pedigree analysis, forensic science, human medical diagnostics and in plant and animal breeding by demonstrating genetic identity, genetic diversity, genomic variation or polymorphism and particularly co-dominant scoring.

Claims

1. A method for demonstrating genetic identity, genetic diversity, genomic variations, polymorphisms, allelic variation or co-dominant scoring within a defined population pool, comprising the steps of:

(a) allowing unlabeled, single-stranded, sample DNA representing total DNA of a sample to hybridize with one or more different sets of oligonucleotide sequences, each oligonucleotide sequence representing a full or an empty integration site of at least one mobile element (ME) and each oligonucleotide sequence further being attached to a defined identifiable location on a solid support;

(b) providing post-hybridization treatments in order to remove sample DNA not fully hybridized to the solid support attached oligonucleotide sequences; and

(c) providing a hybridization product with a recordable label, said label allowing hybridization pattern to be recorded and thereby the presence or absence of a full or an empty integration site of at least one mobile element (ME) to be scored.

2. The method according to claim 1, wherein for co-dominant scoring at least one set of paired or parallel oligonucleotide sequences is provided for each homologue to be scored in the population pool.

3. The method according to claim 1, wherein the hybridization pattern recorded for oligonucleotide sequences representing a full or an empty integration site is used for co-dominant scoring.

4. The method according to claim 1, wherein hybridization takes place under conditions which allow the unlabeled, single-stranded sample DNA to anneal to the oligonucleotide sequences being attached to the solid support.

5. The method according to claim 1, wherein the post-hybridization treatments are carried out under conditions releasing all sample DNA which has not fully hybridized with the oligonucleotide sequences being attached to the solid support.

6. The method according to claim 1, wherein the hybridization pattern is recorded by providing the sample DNA after the hybridization and post-hybridization treatments with a recordable label.

7. The method according to claim 1, wherein the sample DNA after the hybridization and post-hybridization treatments is released from the solid support and subsequently hybridized with labelled oligonucleotide sequences fully corresponding to each of the oligonucleotide sequences which were attached to the solid support.

8. The method according to claim 1, wherein the recording is reversible and the solid support is returned to its original state for reuse.

9. The method according to claim 1, wherein the steps including hybridization, post-hybridization treatment, recording and scoring are automated.

10. The method according to claim 1, wherein the sample DNA is fragmented.

11. The method according to claim 10 wherein hybridization takes place under conditions which allow the unlabelled, single-stranded, fragmented sample DNA to anneal to the oligonucleotide sequences being attached to the solid support.

12. The method according to claim 1, wherein the one or more different sets of oligonucleotide sequences are paired.

13. The method according to claim 1 wherein the one or more different sets of oligonucleotide sequences are parallel.

14. The method according to claim 1, comprising the steps of:

(a) providing a solid support comprising more than one sets of oligonucleotide sequences, each set further comprising at least one oligonucleotide sequence representing a full integration site and one oligonucleotide sequence representing an empty integration site;

(b) shearing the sample DNA representing the total DNA with physical, mechanical or enzymatic means in order to obtain the mobile elements (MEs) onto different pieces of DNA;

(c) rendering said sheared sample DNA single-stranded and allowing said single-stranded sample DNA fragments to hybridize with the single-stranded oligonucleotide sequences attached to the solid support;

(d) providing post-hybridization treatments including removal of single-stranded sample DNA which is not fully hybridized to the oligonucleotide sequences attached to the solid support using washing treatment at different stringencies to remove single-stranded sample DNA fragments not fully corresponding to the attached polynucleotide sequences;

(e) recording a hybridization pattern for each set of oligonucleotide sequences using any method capable of demonstrating the hybridization;

(f) scoring the recordable hybridization pattern wherein the presence of hybridization with a solid support attached oligonucleotide sequence representing a full integration site indicates a presence of at least one mobile element (ME), the presence of hybridization with a solid support attached oligonucleotide sequence representing an empty integration site indicates an absence of a mobile element (ME) in a corresponding integration site, and the absence of hybridization indicates that the integration site is lacking.

15. The method according to claim 14, wherein the post-hybridization treatments include digestion treatments.

16. The method according to claim 1 comprising the steps of:

(a) providing a solid support comprising more than one sets of oligonucleotide sequences, each set further comprising at least one oligonucleotide sequence representing a full integration site and one oligonucleotide sequence representing an empty integration site;

(b) rendering said sheared sample DNA single-stranded and allowing said single-stranded sample DNA fragments to hybridize with the single-stranded oligonucleotide sequences attached to the solid support;

(c) providing post-hybridization treatments including removal of single-stranded sample DNA which is not fully hybridized to the oligonucleotide sequences attached to the solid support using washing treatment at different stringencies to remove single-stranded sample DNA fragments not fully corresponding to the attached polynucleotide sequences;

(d) recording a hybridization pattern for each set of oligonucleotide sequences using any method capable of demonstrating the hybridization;

(e) scoring the recordable hybridization pattern wherein the presence of hybridization with a solid support attached oligonucleotide sequence representing a full integration site indicates a presence of at least one mobile element (ME), the presence of hybridization with a solid support attached oligonucleotide sequence representing an empty integration site indicates an absence of a mobile element (ME) in a corresponding integration site, and the absence of hybridization indicates that the integration site is lacking.

17. The method according to claim 16, wherein the post-hybridization treatments include digestion treatments.

18. The method according to the claim 1 comprising the steps of:

(a) providing a solid support comprising more than one sets of oligonucleotide sequences, each set further comprising at least one oligonucleotide sequence representing a full integration site and one oligonucleotide sequence representing an empty integration site;

(b) shearing the sample DNA representing the total DNA with physical, mechanical or enzymatic means in order to obtain the mobile elements (MEs) onto different pieces of DNA;

(c) rendering said sheared sample DNA single-stranded and allowing said single-stranded sample DNA fragments to hybridize with the single-stranded oligonucleotide sequences attached to the solid support;

(d) recording a hybridization pattern for each set of oligonucleotide sequences using any method capable of demonstrating the hybridization;

(e) scoring the recordable hybridization pattern wherein the presence of hybridization with a solid support attached oligonucleotide sequence representing a full integration site indicates a presence of at least one mobile element (ME), the presence of hybridization with a solid support attached oligonucleotide sequence representing an empty integration site indicates an absence of a mobile element (ME) in a corresponding integration site, and the absence of hybridization indicates that the integration site is lacking.

19. The method according to claim 1, comprising the steps of:

(a) providing a solid support comprising more than one sets of oligonucleotide sequences, each set further comprising at least one oligonucleotide sequence representing a full integration site and one oligonucleotide sequence representing an empty integration site;

(b) rendering said sheared sample DNA single-stranded and allowing said single-stranded sample DNA fragments to hybridize with the single-stranded oligonucleotide sequences attached to the solid support;

(c) recording a hybridization pattern for each set of oligonucleotide sequences using any method capable of demonstrating the hybridization;

(d) scoring the recordable hybridization pattern wherein the presence of hybridization with a solid support attached oligonucleotide sequence representing a full integration site indicates a presence of at least one mobile element (ME), the presence of hybridization with a solid support attached oligonucleotide sequence representing an empty integration site indicates an absence of a mobile element (ME) in a corresponding integration site, and the absence of hybridization indicates that the integration site is lacking.

20. The method according to claim 1, wherein each oligonucleotide sequence attached to the solid support represents a junction in at least one full or one corresponding empty integration site of a mobile element (ME);

an oligonucleotide sequence representing a junction in a full integration site further comprising a first and a second distinct sequence region, the first distinct sequence region being a region flanking the mobile element (ME) and the second distinct sequence region being a terminal end of the mobile element (ME); and

an oligonucleotide sequence representing a junction in an empty integration site further comprising two distinct sequence regions each composed of flanking regions surrounding the integration site of the mobile element (ME).

21. The method according to claim 20, wherein the first distinct sequence region is longer than the second distinct sequence region.

22. The method according to claim 20, wherein the first and the second distinct sequence regions of the oligonucleotide sequence representing a junction in a full integration site are of equal length.

23. The method according to claim 20, wherein the first and the second distinct sequence regions of the oligonucleotide sequence representing a junction in a full integration site are of varying length.

36. The method according to claim 1 for distinguishing any organism differing in at least one integration site of at least one mobile element (ME) integration site in any given genomic position.

38. The method according to claim 1 for genotyping, phylogenetic studies, parenthood determinations, forensic science, human medical diagnostics, haplotyping, and pedigree analysis and in plant and animal breeding by demonstrating genetic identity, genetic diversity, genomic variation or polymorphism and particularly co-dominant scoring.

40. The method according to claim 1 for assured and accelerated breeding.

24. A method for demonstrating genetic identity, genetic diversity, genomic variations, polymorphisms, allelic variation or co-dominant scoring within a defined population pool, comprising the steps of:

a) allowing unlabeled, single-stranded, sample DNA representing total DNA of a sample to hybridize with one or more different sets of oligonucleotide sequences, each oligonucleotide sequence representing a full or an empty integration site of at least one mobile element (ME) and each oligonucleotide sequence further being attached to a defined identifiable location on a solid support;

b) providing a hybridization product with a recordable label, said label allowing hybridization pattern to be recorded and thereby the presence or absence of a full or an empty integration site of at least one mobile element (ME) to be scored.

25. A test kit for demonstrating genetic identity, genetic diversity, genomic variations, polymorphisms, allelic variation or co-dominant scoring in a population pool, said test kit further comprising more than one set of single-stranded oligonucleotide sequences, each oligonucleotide sequence representing a junction in at least one full or one corresponding empty integration site, the oligonucleotide sequence representing junction in a full integration site further comprising a first and a second distinct sequence region, the first distinct sequence region being a flanking region of a mobile element (ME) and the second distinct sequence region being a terminal end of said mobile element (ME) and a oligonucleotide sequence representing the corresponding empty integration site further comprising two flanking regions surrounding said mobile element (ME).

26. The test kit according to claim 25, wherein the first distinct sequence is longer than the second distinct sequence region.

27. The test kit according to claim 25, wherein at least one set of oligonucleotide sequences is provided for each homologue to be scored present in the population pool.

28. The test kit according to claim 25, wherein the oligonucleotide sequences are paired.

29. The test kit according to claim 25, wherein the oligonucleotide sequences are parallel.

30. The test kit according to claim 25, wherein the test kit further comprises at least one pair of oligonucleotide sequences attached to a solid support, one of said oligonucleotide sequences representing a full site and one of said oligonucleotide sequences representing an empty site thereby allowing co-dominant scoring.

31. The test kit according to claim 25, wherein the solid support comprises at least one membrane, filter, slide, plate, chip, dish or microwell composed of material selected from the group consisting of glass, plastics, nitrocellulose, nylon, polyacrylic acids and silicons.

32. The test kit according claim 25, wherein the test kit comprises elements selected from a group consisting of reagents, labels, washing buffers, end protecting reagents and instructions for use.

33. The test kit according claim 25, wherein the oligonucleotide sequences have a size allowing a formation of a stable hybridization product between the solid support attached oligonucleotide and the sample DNA.

34. The test kit according to claim 25, wherein the oligonucleotide sequences are end-protected.

35. The test kit according to claim 25, wherein the recording treatments are reversible allowing the solid support to be returned to its original state for reuse.

37. The test kit according to claim 25 for distinguishing any organism differing in at least one integration site of at least one mobile element (ME) integration site in any given genomic position.

39. The test kit according to claim 25 for genotyping, phylogenetic studies, parenthood determinations, forensic science, human medical diagnostics, haplotyping, and pedigree analysis and in plant and animal breeding by demonstrating genetic identity, genetic diversity, genomic variation or polymorphism and particularly co-dominant scoring.

41. The test kit according to claim 25 for assured and accelerated breeding.