METHODS AND COMPOSITIONS FOR ISOLATING NUCLEIC ACID SEQUENCE VARIANTS

• US 20090004665A1
• Filed: 06/27/2008
• Published: 01/01/2009
• Est. Priority Date: 06/29/2007
• Status: Active Grant

• Current Assignee: Agency For Science Technology and Research
• Original Assignee: Population Genetics Technologies Ltd.
• Inventors: Brenner, Sydney
• Granted Patent: US 7,635,566 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer

Abstract

The invention is drawn to isolating sequence variants of a genetic locus of interest using a modified iterative primer extension method. The nucleic acids analyzed are generally single stranded and have a reference sequence which is used as a basis for performing iterative single nucleotide extension reactions from a hybridized polymerization primer. The iterative polymerization reactions are configured such that polymerization of the strand will continue if the sequence of the nucleic acid being analyzed matches the reference sequence, whereas polymerization will be terminated if the nucleic acid being analyzed does not match the reference sequence. Nucleic acid strands that have mutations can be isolated using a variety of methods and sequenced to determine the precise identity of the mutation/polymorphism. By performing the method on both strands of the nucleic acid being analyzed, virtually all possible mutations can be identified.

Claims

1. A method of isolating nucleic acid molecules having a nucleotide sequence different from a reference sequence in a locus of interest, said method comprising:

(i) providing a sample comprising nucleic acid molecules having a locus of interest;

(ii) annealing a polymerization primer to said nucleic acid molecules immediately upstream of said locus of interest;

(iii) contacting said polymerization primer-annealed nucleic acid molecules with a polymerization reagent pool under nucleic acid polymerization conditions, wherein said polymerization reagent pool comprises a nucleotide polymerase and two or more nucleotide bases, wherein said two or more nucleotide bases are selected based on the identity of the next two different nucleotide bases predicted to be incorporated at unoccupied sites downstream of said polymerization primer as determined by a reference sequence for said locus of interest; and

(iv) isolating nucleic acid molecules in which said two or more nucleotides have not been incorporated as predicted based on said reference sequence for said locus of interest, thereby isolating nucleic acid molecules having a nucleotide sequence different from said reference sequence in said locus of interest.

2. The method of claim 1, wherein said sample comprises nucleic acids from a plurality of subjects, wherein nucleic acids from each of said plurality of subjects is tagged with a unique nucleotide tag.

3. The method of claim 2, wherein differences are identified in two or more of said plurality of subjects.

4. The method of claim 2, wherein said method further comprises determining from which of said plurality of subjects said isolated nucleic acid is derived based on said unique nucleotide tag.

5. The method of claim 1, wherein said method further comprises one or both of:

sub-cloning said isolated nucleic acid molecules into a vector and sequencing said isolated nucleic acid molecules.

6. The method of claim 1, wherein said polymerization reagent pool comprises a deoxyribonucleotide and two dideoxyribonucleotides, wherein said deoxyribonucleotide is predicted to be the first of said next two different nucleotide bases predicted to be incorporated at unoccupied sites downstream of said polymerization primer and said two dideoxyribonucleotides are predicted not to be either of said next two different nucleotide bases predicted to be incorporated at unoccupied sites downstream of said polymerization primer.

7. The method of claim 6, wherein said dideoxyribonucleotides are labeled with a first member of a binding pair and said isolating step comprises:

(a) contacting said sample to a second member of said binding pair, and

(b) isolating nucleic acids bound to said second member of said binding pair.

8. The method of claim 7, wherein said binding pair is an avidin/biotin binding pair.

9. The method according to claim 6, wherein each of said two dideoxyribonucleotides are labeled with distinguishing detectable labels.

10. The method according to claim 9, wherein said method further comprises:

screening said isolated nucleic acid molecules for the presence of said distinguishing detectable labels; and

determining how the sequence of said isolated nucleic acid molecules differs from said reference sequence based on said screening.

11. The method of claim 6, wherein said method further comprises immobilizing said nucleic acid molecules having said locus of interest to a substrate, wherein said immobilizing comprises hybridizing said nucleic acid molecules having said locus of interest to a capture primer attached to said substrate, wherein said capture primer binds to the same strand of said nucleic acid molecules as said polymerization primer at a location that is downstream of said polymerization primer, and wherein said isolation step further comprises:

contacting said immobilized nucleic acid molecules under polymerization conditions to a polymerization reagent pool comprising all four deoxyribonucleic acid triphosphates and a strand-displacing nucleotide polymerase; and

isolating nucleic acid molecules that remain bound to said substrate-attached capture primer.

12. The method of claim 1, wherein said method further comprises repeating step (iii) for a predetermined number of cycles.

13. The method of claim 12, wherein said isolating step is performed in each of said predetermined number of cycles.

14. The method of claim 12, wherein said isolating step is performed after completion of all of said predetermined number of cycles.

15. The method of claim 1, wherein multiple nucleic acid molecules having distinct nucleic acid differences from said reference sequence in said locus of interest are isolated.

16. A method of eluting a nucleic acid from a substrate, said method comprising:

(i) obtaining a nucleic acid immobilized on a substrate via hybridization to a capture primer attached to said substrate;

(ii) annealing a polymerization primer to said immobilized nucleic acid, wherein said polymerization primer hybridizes upstream of said capture primer; and

(iii) contacting said polymerization primer-annealed immobilized nucleic acid to a strand-displacing nucleotide polymerase under nucleic acid polymerization conditions;

wherein said strand displacing nucleotide polymerase displaces said capture primer from said nucleic acid, thereby eluting said nucleic acid from said substrate.

17. A method of sorting nucleic acid molecules according to the identity of their corresponding unique nucleotide tags, said method comprising:

(i) contacting a nucleic acid sample comprising at least two nucleic acid molecules each having a unique tag to a first oligonucleotide primer under hybridization conditions, wherein said first oligonucleotide primer is immobilized on a substrate and comprises a sequence complementary to a first region present in said at least two nucleic acid molecules, whereby said at least two nucleic acid molecules are immobilized;

(ii) contacting said immobilized at least two nucleic acid molecules to a second oligonucleotide primer under hybridization conditions, wherein said second primer comprises a sequence complementary to a region in the unique tag of a first of said at least two nucleic acid molecules, wherein said second primer binds to the same strand as said first primer at a location that is 5′

to said first primer; and

(iii) isolating said first of said at least two nucleic acid molecules by contacting said immobilized at least two nucleic acid molecules to a displacing nucleotide polymerase under nucleic acid polymerization conditions, wherein extension of said second primer displaces said first of said at least two nucleic acid molecules from said first immobilized primer;

wherein said at least two uniquely-tagged nucleic acid molecules are sorted according to the identity of their corresponding unique nucleotide tags.

18. The method of claim 17, wherein said method further comprises isolating a second of said at least two nucleic acid molecules by repeating steps (ii) and (iii) using a third oligonucleotide primer, wherein said third primer comprises a sequence complementary to a region in the unique tag of a second of said at least two nucleic acid molecules, wherein said third primer binds to the same strand as said first primer at a location that is 5′

to said first primer.

19. A polymerization reagent pool set comprising a plurality polymerization reagent pools, wherein each of said plurality of polymerization reagent pools comprises:

(i) a nucleotide polymerase;

(ii) a deoxyribonucleotide base; and

(iii) a dideoxyribonucleotides nucleotide base different from said deoxyribonucleotide base;

wherein each of said plurality of polymerization reagent pools has a unique combination of said deoxyribonucleotide and said dideoxyribonucleotide base, and wherein said polymerization reagent pool set is designed for isolating nucleic acid molecules having a nucleotide sequence different from a reference sequence in a locus of interest according to the method of claims.

20. The polymerization reagent pool set of claim 19, wherein said two dideoxyribonucleotides are labeled with distinguishing detectable labels.