Compositions, Kits and Methods for Isolating Target Polynucleotides
AssigneeDNAe Diagnostics Limited
InventorsNorm Nelson, David Wooldridge
Life of this patent
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Abstract
Provided herein are oligomers, compositions, kits, and methods for capturing target polynucleotides, e.g., for downstream applications such as amplification, library preparation, or sequencing. In some embodiments, a capture oligomer is provided or used that comprises a capture sequence that is annealed to a complement that prevents capture until the complement is displaced in a target-polynucleotide dependent manner. In some embodiments, an amount of target polynucleotide is captured that is less than or equal to a predetermined amount.
Claims8
-30. (canceled)
A combination comprising a capture oligomer and a complementary oligomer, wherein:
- (a) the capture oligomer comprises, in the 5′ to 3′ direction:
- a capture sequence comprising first and second portions,
- an internal extension blocker,
- a spacer sequence comprising first and second portions, and
- a target-hybridizing sequence;
- and
- (b) the complementary oligomer comprises, in the 3′ to 5′ direction:
- a complement of the second portion of the capture sequence, and
- a complement of at least the first portion of the spacer sequence, wherein the complement of the second portion of the capture sequence and the complement of the at least first portion of the spacer sequence are configured to anneal simultaneously to the capture oligomer in the absence of a complement of the spacer sequence.
A method of capturing a target polynucleotide from a composition, the method comprising:
- contacting the composition with the combination of claim 1, wherein the target-hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site comprising the 3′ end of the target polynucleotide;
- extending the 3′ end of the target polynucleotide with a DNA polymerase with strand-displacement activity, thereby forming a complement of the spacer sequence, which is annealed to the capture oligomer, such that the complementary oligomer is displaced to an extent sufficient for the capture sequence of the capture oligomer to be available for binding;
- contacting the capture sequence of the capture oligomer with a secondary capture reagent comprising a complement of the capture sequence and (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent;
- and
- isolating the complex from the composition, thereby capturing the target polynucleotide.
Dependencies are inferred from the claim text.