Genetic analysis by sequence-specific sorting

• US 20060177832A1
• Filed: 06/30/2005
• Published: 08/10/2006
• Est. Priority Date: 02/10/2005
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research (Government of Singapore)
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Granted Patent: US 7,407,757 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

The invention provides methods for sorting polynucleotides from a population based on predetermined sequence characteristics. In one aspect, the method of the invention is carried out by extending a primer annealed polynucleotides having predetermined sequence characteristics to incorporate a predetermined terminator having a capture moiety, capturing polynucleotides having extended primers by a capture agent that specifically binds to the capture moiety, and melting the captured polynucleotides from the extended primers to form a subpopulation of polynucleotides having the predetermined sequence characteristics. In another aspect, the method of the invention is carried out on a population of tagged polynucleotides so that after a subpopulation is selected, the members of the subpopulation may be simultaneously analyzed using the unique tags on the polynucleotides to convey analytical information to a hybridization array for a readout.

Claims

1. A method of sorting polynucleotides having predetermined sequence characteristics, the method comprising the steps of:

extending a primer annealed polynucleotides having predetermined sequence characteristics to incorporate a predetermined terminator having a capture moiety;

capturing polynucleotides having extended primers by a capture agent that specifically binds to the capture moiety; and

melting the captured polynucleotides from the extended primers.

2. The method of claim 1 wherein said predetermined terminator comprises a mixture of four kinds of terminator each complementary to a different nucleoside and wherein each kind of terminator has a different capture moiety.

3. The method of claim 1 further including the step of separating said polynucleotides into different mixtures prior to said step of extending so that in each different mixture said primer is extended with a terminator complementary to a different nucleoside.

4. The method of claim 3 wherein said capture moiety is a biotin and wherein said terminators are each dideoxynucleotides.

5. A method of producing a subpopulation of polynucleotides having a complexity less than that of a parent population, the method comprising the steps of:

annealing a primer to polynucleotides of a parent population to form primer-polynucleotide duplexes;

extending the primer to incorporate a predetermined terminator having a capture moiety;

separating the primer-polynucleotide duplexes having an extended primer from the parent population by specifically binding the capture moiety of the predetermined terminator to a capture agent attached to a solid phase support;

melting the primer-polynucleotide duplexes to form a subpopulation of polynucleotides having a complexity less than that of the parent population.

6. The method of claim 5 wherein said predetermined terminator comprises a mixture of four kinds of terminator each complementary to a different nucleoside and wherein each kind of terminator has a different capture moiety.

7. The method of claim 5 further including the step of separating said polynucleotides of said parent population into different mixtures prior to said step of extending so that in each different mixture said primer is extended with a terminator complementary to a different nucleoside.

8. The method of claim 7 wherein said capture moiety is a biotin and wherein said terminators are each dideoxynucleotides.

9. A method of producing a population of polynucleotides having a desired complexity less than that of a parent population, the method comprising the steps of:

(a) annealing a primer to polynucleotides of a parent population to form primer-polynucleotide duplexes;

(b) extending the primer to incorporate a predetermined terminator having a capture moiety;

(c) separating the primer-polynucleotide duplexes having an extended primer from the parent population by specifically binding the capture moiety of the predetermined terminator to a capture agent attached to a solid phase support;

(d) melting the primer-polynucleotide duplexes to form a selected population of polynucleotides having a complexity less than that of the parent population, the selected population forming a parent population for subsequent steps;

(e) repeating steps (a) through (d) until a selected population of the desired complexity is obtained.

10. The method of claim 9 wherein said predetermined terminator comprises a mixture of four kinds of terminator each complementary to a different nucleoside and wherein each kind of terminator has a different capture moiety.

11. The method of claim 9 wherein said step of annealing further includes separating said polynucleotides of said parent population into different mixtures prior to said step of extending so that in each different mixture said primer is extended with a terminator complementary to a different nucleoside.

12. The method of claim 11 wherein said capture moiety is a biotin and wherein said terminators are each dideoxynucleotides.

13. The method of claim 9 further comprising a step of replicating said selected population after said step of melting.

14. The method of claim 13 wherein during each said step of repeating steps (a) through (d), said primer anneals to a different primer binding site on said polynucleotides of said parent population or said selected population.

15. The method of claim 14 wherein in each successive step of repeating steps (a) through (d), said different primer binding site is shifted along said polynucleotides at least one nucleotide in a primer extension direction.

16. The method of claim 14 wherein in each successive step of repeating steps (a) through (d), said different primer binding site is at a different and non-overlapping locus of said polynucleotides.

17. The method of claim 16 wherein said different and non-overlapping locus is adjacent to and upstream of a single nucleotide polymorphism site.

18. A method of determining polynucleotides that have sequences that vary from that of a reference sequence, the method comprising the steps of:

(a) annealing a primer to polynucleotides of a parent population to form primer-polynucleotide duplexes;

(b) extending the primer in the presence of at least one predetermined terminator having a capture moiety, the predetermined terminator being non-complementary with the reference sequence, so that after extension primer-polynucleotide complexes that contain a polynucleotide having a sequence different from that of the reference sequence have capture moieties;

(c) separating the primer-polynucleotide duplexes having an extended primer with a capture moiety from the parent population by specifically binding the capture moiety of the predetermined terminator to a capture agent;

(d) melting captured primer-polynucleotide duplexes to form a selected population of polynucleotides having sequences different from that of the reference sequence;

(e) shortening polynucleotides of the parent population by from 1 to 20 nucleotides to form a new parent population;

(f) repeating steps (a) through (e) until all the polynucleotides of the parent population have been sorted from polynucleotides having sequences identical to the reference sequence.

19. The method of claim 18 wherein said step of shortening includes shortening said polynucleotides of said parent population by one nucleotide.

20. The method of claim 19 wherein said step of annealing further includes separating said polynucleotides of said parent population into different mixtures prior to said step of extending so that in each different mixture said primer is extended with a terminator complementary to a different nucleoside.

21. The method of claim 20 wherein said capture moiety is a biotin and wherein said terminators are each dideoxynucleotides.

22. A method of determining a frequency of a nucleotide at a predetermined locus in a population of genomes, the method comprising the steps of:

separately generating fragments of each genome of the population;

attaching a unique oligonucleotide tag to each genome;

selecting fragments from each genome that contains the predetermined locus;

generating a labeled oligonucleotide tag from each unique oligonucleotide tag, the labeled oligonucleotide tag generating a signal indicative of the nucleotide at the predetermined locus; and

determining the frequency of the nucleotide at the predetermined locus by detecting the signals generated by the labeled oligonucleotide tags specifically hybridized with their respective tag complements, the respective tag complements being attached in spatially discrete regions on the one or more solid phase supports.

23. The method of claim 22 wherein said oligonucleotide tag comprises a concatenate of words each having a position such that the word of each position identifies a mixture of fragments from which said oligonucleotide tag and its associated fragment are derived.

24. The method of claim 23 wherein said one or more solid supports is a microarray.