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US 12,188,079 B2Granted
Determining the sequence of a double-stranded target nucleic acid by employing a terminal transferase, forming a linear joint molecule, and sequencing in one direction
AssigneeRoche Sequencing Solutions, Inc.
InventorsRui Chen, Toumy Guettouche, Loida Navarro, Aaron Richardson
Life of this patent
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Abstract
The invention is a novel method of making and using a template for nucleic acid sequencing. The templates include circular and linear templates with symmetric and asymmetric adaptors. The methods include utilizing the templates in an asymmetric fashion.
Claims12
A method of determining a sequence of a double-stranded target nucleic acid in a sample, comprising:
- (a) contacting the sample comprising the double-stranded target nucleic acid, wherein the double-stranded target nucleic acid has two 5′-ends and two extendable 3′-ends, with a terminal transferase and a single extendable nucleotide species, and providing means for controlling nucleotide incorporation by the terminal transferase;
- (b) extending the two extendable 3′-ends by incorporating multiple units of the single nucleotide, thereby forming a linear joint molecule comprising the target nucleic acid and having:
- (i) a double-stranded region, (ii) a proximal single-stranded homopolymer region, and (iii) a distal single-stranded homopolymer region;
- (c) contacting the sample with a limiting concentration of capture oligonucleotide tethered to a solid support, wherein the capture oligonucleotide is complementary to the proximal and distal single-stranded homopolymer regions, thereby capturing the linear joint molecule on the solid support;
- (d) contacting the sample with an oligonucleotide primer complementary to the proximal and distal single-stranded homopolymer regions, thereby hybridizing the primer to the distal single-stranded homopolymer region;
- and
- (e) extending the primer with a sequencing polymerase, thereby determining the sequence of the double-stranded target nucleic acid.
4 more elements
A composition for determining a sequence of a double-stranded target nucleic acid comprising:
- (a) a linear joint molecule comprising the target nucleic acid and having a double-stranded region and proximal and distal single-stranded homopolymer regions;
- (b) a capture oligonucleotide tethered to a solid support and hybridized to the proximal single-stranded homopolymer region, thereby capturing the linear molecule on the solid support;
- (c) an oligonucleotide primer hybridized to the distal single-stranded homopolymer region;
- and
- (d) a sequencing polymerase.
2 more elements
A method of determining a sequence of a double-stranded target nucleic acid in a sample, comprising:
- (a) contacting the sample comprising the double-stranded target nucleic acid, wherein the double-stranded target nucleic acid has two 5′-ends and two extendable 3′-ends, with a terminal transferase and a single extendable nucleotide species, and providing means for controlling nucleotide incorporation by the terminal transferase;
- (b) extending the two extendable 3′-ends by incorporating multiple units of the single nucleotide, thereby forming a linear joint molecule comprising the target nucleic acid and having:
- (i) a double-stranded region, (ii) a proximal single-stranded homopolymer region, and (iii) a distal single-stranded homopolymer region, wherein the hompolymer regions of the proximal single-stranded hompolymer region and the distal single-stranded hompolymer region are different;
- (c) contacting the sample with a limiting concentration of capture oligonucleotide tethered to a solid support, wherein the capture oligonucleotide is complementary to the proximal single-stranded homopolymer region, thereby capturing the linear joint molecule on the solid support;
- (d) contacting the sample with an oligonucleotide primer complementary to the distal single-stranded homopolymer region, thereby hybridizing the primer to the distal single-stranded homopolymer region;
- and
- (e) extending the primer with a sequencing polymerase, thereby determining the sequence of the double-stranded target nucleic acid.
4 more elements
Dependencies are inferred from the claim text.