DNA-based methods of geochemical prospecting

• US 8,071,295 B2
• Filed: 06/25/2003
• Issued: 12/06/2011
• Est. Priority Date: 04/10/2000
• Status: Expired due to Fees

• Current Assignee: Taxon Biosciences, Inc. (Evogene Ltd)
• Original Assignee: Matthew Ashby
• Inventors: Ashby, Matthew
• Primary Examiner(s): Strzelecka, Teresa E
• Application Number:
  US10/607,077
• Publication Number:
  US 20040110183A1
• Time in Patent Office: 3,086 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 culture-independent method of determining the abundance of an environmental parameter in an environmental sample, said parameter not including bacteria, comprising the steps of:

a. providing a first plurality of environmental samples at least some of which samples contain the environmental parameter;

b. isolating a plurality of genomic DNAs from each of the samples provided in step a;

c. isolating a plurality of 16S rRNA gene segments from each plurality of genomic DNAs isolated in step b;

d. determining the abundance of each of said 16S rRNA gene segments in each plurality of 16S rRNA gene segments isolated in step c;

e. determining the abundance of the environmental parameter in each of the samples provided in step a;

f. correlating the abundance of each 16S rRNA gene segment determined in step d with the abundance of the environmental parameter determined in step e;

g. selecting at least one 16S rRNA gene segment whose abundance correlates to the abundance of said environmental parameter, as determined in step f;

h. providing an environmental sample set of at least one environmental sample;

i. isolating a plurality of genomic DNAs from each environmental sample of the environmental sample set provided in step h;

j. determining the abundance of said 16S rRNA gene segment selected in step g in each plurality of genomic DNAs isolated in step i; and

k. inferring the abundance of the environmental parameter in each environmental sample of the environmental sample set provided in step h based upon the abundance of said 16S rRNA gene determined in step j in each environmental sample of the environmental sample set provided in step h.

2. The method according to claim 1, wherein the correlation in step g is expressed by an r-value of 1.

3. The method according to claim 1, wherein the correlation in step g is expressed by an r-value of 0.8 to 0.99.

4. The method according to claim 1, wherein the correlation in step g is expressed by an r-value of 0.5 to 0.7.

5. The method according to claim 1, wherein the environmental parameter is subsurface oil or natural gas.

6. The method of claim 1, wherein the determination in step j, of the abundance of said 16S rRNA gene segment, is done using PCR.

7. The method of claim 1, wherein the determination in step j, of the abundance of said 16S rRNA gene segment, is done using a hybridization assay.

17. The method according to claim 1, 8 or 13, wherein the environmental parameter is a mineral deposit.

8. A culture-independent method of identifying an indicator for an environmental parameter, said parameter not including bacteria, said method comprising the steps of:

a. providing a plurality of environmental samples at least some of which samples contain the environmental parameter;

b. isolating a plurality of genomic DNAs from each of the samples provided in step a;

c. isolating a plurality of 16S rRNA gene segments from each plurality of genomic DNAs isolated in step b;

d. determining the abundance of each of said 16S rRNA gene segments in each plurality of 16S rRNA gene segments isolated in step c;

e. determining the abundance of the environmental parameter in each of the samples provided in step a;

f. correlating the abundance of each 16S rRNA gene segment determined in step d with the abundance of the environmental parameter determined in step e;

g. selecting at least one 16S rRNA gene segment whose abundance correlates to the abundance of said environmental parameter, as determined in step f;

h. designating said 16S rRNA gene segment as the indicator.

9. The method according to claim 8, wherein the correlation in step g is expressed by an r-value of 1.

10. The method according to claim 8, wherein the correlation in step g is expressed by an r-value of 0.8 to 0.99.

11. The method according to claim 8, wherein the correlation in step g is expressed by an r-value of 0.5 to 0.7.

12. The method according to claim 8, wherein the environmental parameter is subsurface oil or natural gas.

13. A culture-independent method of determining the abundance of an environmental parameter in an environmental sample, said parameter not including bacteria, comprising the steps of:

a. providing at least one environmental sample;

b. isolating a plurality of genomic DNAs from said environmental sample(s);

c. determining the abundance of the indicator identified in the method of claim 8 in the plurality of genomic DNAs isolated in step b; and

d. inferring the abundance of the environmental parameter in said sample(s) based upon the abundance of said indicator in said sample(s).

14. The method according to claim 13, wherein the environmental parameter is subsurface oil or natural gas.

15. The method of claim 13, wherein the abundance of said indicator in said plurality of genomic DNAs is determined using PCR.

16. The method of claim 13, wherein the abundance of said indicator in said plurality of genomic DNAs is determined using a hybridization assay.