Genetic analysis by sequence-specific sorting
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.
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.