Methods for the survey and genetic analysis of populations

• US 6,613,520 B2
• Filed: 04/10/2001
• Issued: 09/02/2003
• Est. Priority Date: 04/10/2000
• Status: Expired due to Term

• Current Assignee: Taxon Biosciences, Inc. (Evogene Ltd)
• Original Assignee: Matthew Ashby
• Inventors: Ashby, Matthew
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): STRZELECKA, TERESA E
• Application Number:
  US09/829,855
• Publication Number:
  US 20020065609A1
• Time in Patent Office: 875 Days
• Field of Search: 435/6, 435/91.2, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 20/00 ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
• CPC Class Codes: G16B 20/40 Population genetics; Linkage disequilibrium
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly
• CPC Class Codes: Y10T 436/21 Hydrocarbon

Abstract

The present invention relates to methods for performing surveys of the genetic diversity of a population. The invention also relates to methods for performing genetic analyses of a population. The invention further relates to methods for the creation of databases comprising the survey information and the databases created by these methods. The invention also relates to methods for analyzing the information to correlate the presence of nucleic acid markers with desired parameters in a sample. These methods have application in the fields of geochemical exploration, agriculture, bioremediation, environmental analysis, clinical microbiology, forensic science and medicine.

Claims

1. A method to determine the genetic diversity of a population by determining the DNA sequence of a targeted polymorphic region from one gene that is common to all, or the majority of all, members of the population, comprising the steps of:

a) providing a sample containing a population that comprises at least one genomic DNA molecule containing regions of high sequence conservation flanking a region of polymorphic sequence;

b) isolating the genomic DNA molecule from the sample;

c) performing PCR on the genomic DNA molecule, to amplify a subregion of the region of polymorphic sequence, using an upstream primer comprising a binding site for a solid support and a downstream primer to produce an amplified fragment, wherein the upstream primer and the downstream primer hybridize to the regions of high sequence conservation flanking the region of polymorphic sequence;

d) cleaving the amplified fragment with a restriction enzyme that cleaves the amplified fragment at a site adjacent to the region of polymorphic sequence;

e) binding the cleaved amplified fragment to a solid support to form an immobilized product;

f) splitting the immobilized product into two pools;

g) attaching a double-stranded linker to the immobilized product of each pool, wherein the linker comprises a Type IIS restriction enzyme site;

h) digesting the immobilized product with a Type IIS restriction enzyme to form a tag;

i) ligating the tag to a second tag to form a ditag;

j) amplifying the ditag by PCR;

k) cleaving the ditag with the restriction enzyme of step d) and ligating the ditag to a second ditag to form a concatemer; and

l) determining a DNA sequence of the concatemer, wherein the sequence of the concatemer is a sequence of a multitude of markers, which provides a measure of the genetic diversity of a population, thereby allowing for the determination of the genetic diversity of a population.

2. The method according to claim 1, further comprising the step of digesting the tag generated in step g) with T4 DNA polymerase to create blunt ends.

3. The method according to claim 1, further comprising the step of amplifying the ditag a second time after step i).

4. The method according to claim 1, wherein the population is selected from the group consisting of a microbial population, a viral population and an immune cell population.

5. The method according to claim 4, wherein the microbial population is from a sample from a site for petroleum or natural gas exploration.

6. The method according to claim 4, wherein the microbial population is from a sample from a site of potential oil or gas reserves.

7. The method according to claim 4, wherein the microbial population is from a sample from a site for mineral exploration.

8. The method according to claim 4, wherein the microbial population is from a sample from an agricultural field.

9. The method according to claim 4, wherein the microbial population is from an individual.

10. The method according to claim 9, wherein the individual is suspected of having a bacterial of fungal infection.

11. The method according to claim 4, wherein the microbial population is from a sample from a bioremediation site.

12. The method according to claim 4, wherein the immune cell population is from an individual.

13. The method according to claim 12, wherein the individual is suffering from a disease selected from the group consisting of autoimmune disease, graft v. host disease, lymphoma, leukemia, Hodgkin'"'"'s disease, cancer, atherosclerosis and psoriasis.

14. The method according to claim 4, wherein the microbial population is from a sample of an insect or a parasite.

15. The method according to claim 1, further comprising the step of cloning the concatemer into a sequencing vector.

16. The method according to claim 9, wherein the individual is a human.

17. The method according to claim 10, wherein the individual is a human.

18. The method according to claim 12, wherein the individual is a human.

19. The method according to claim 13, wherein the individual is a human.