Genetic analysis by sequence-specific sorting

• US 7,217,522 B2
• Filed: 02/10/2005
• Issued: 05/15/2007
• Est. Priority Date: 02/12/2004
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Campass Genetics LLC
• Inventors: Brenner, Sydney
• Primary Examiner(s): Martinell; James
• Application Number:
  US11/055,187
• Publication Number:
  US 20050181408A1
• Time in Patent Office: 824 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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 to form a subpopulation of reduced complexity, the method comprising the steps of:

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

capturing polynucleotides having an extended primer by a capture agent that specifically binds to the capture moiety;

melting the captured polynucleotides from the extended primer; and

isolating the melted polynucleotides;

thereby recovering an isolated subpopulation of reduced complexity.

2. 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; and

melting the primer-polynucleotide duplexes, thereby recovering an isolated subpopulation of polynucleotides having a complexity less than that of the parent population.

3. 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 polynucleotides from the primer-polynucleotide duplexes attached to the solid phase support, thereby recovering 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; and

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

4. The method of claim 3 further comprising a step of replicating said selected population after said step of melting.

5. The method of claim 4 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.

6. The method of claim 5 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.

7. The method of claim 5 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.

8. The method of claim 7 wherein said different and non-overlapping locus is adjacent to and upstream of a single nucleotide polymorphism site.