Methods and compositions for isolating asymmetric nucleic acid complexes
AssigneePacific Biosciences of California, Inc.
InventorsLei Sun, Sassan Sheikholeslami, Natasha Popovich, David Christopher Scherer
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Abstract
The present disclosure provides improved methods for isolating asymmetrically-primed and/or asymmetrically-tagged nucleic acid complexes that find use in downstream analytical analyses, including sequence analysis. Compositions comprising such complexes and kits and systems for generating such complexes are also provided.
Claims21
The method of claim 42, wherein the providing step comprises providing
- a mixture of symmetrically-primed nucleic acid templates and asymmetrically-primed nucleic acid templates, wherein each symmetrically-primed nucleic acid template comprises capture primers hybridized to both terminal hairpin adapters or synthesis primers hybridized to both terminal hairpin adapters, and wherein each asymmetrically-primed nucleic acid template comprises a capture primer hybridized to the hairpin adapter at one end and a synthesis primer hybridized to the hairpin adapter at the opposite end;
- and
- wherein the eluting step comprises maintaining the immobilized templates and the nucleic acid polymerase under conditions that promote nucleic acid synthesis from the synthesis primer, thereby eluting asymmetrically-primed nucleic acid templates from the solid support.
The method of claim 42, wherein nucleic acid synthesis is allowed to continue after elution of the nucleic acid templates from the solid support.
The method of claim 45, wherein the capture region on the capture primer is a binding partner for the immobilized binding moiety on the solid support.
10-22. (canceled)
The method of 42, wherein the strand displacing polymerase is a Φ29 DNA polymerase, a homolog of a Φ29 DNA polymerase, a modified version of a Φ29 DNA polymerase, or a modified version of a homolog of a Φ29 DNA polymerase.
The method of claim 42, wherein the isolated nucleic acid templates are subjected to sequence analysis.
29-41. (canceled)
A method for isolating nucleic acid templates comprising:
- providing nucleic acid templates that comprise a double-stranded insert region with hairpin adapters at both ends, wherein at least a portion of the templates comprise a capture primer is hybridized to the hairpin adapter at one end;
- immobilizing the capture-primer hybridized templates to a solid support through the capture primer;
- eluting the templates from the solid support by maintaining the immobilized templates and a nucleic acid polymerase having strand displacement activity under conditions that promote nucleic acid synthesis;
- and
- collecting the templates eluted from the solid support by the strand displacement activity of the nucleic acid polymerase.
Dependencies are inferred from the claim text.