Phospholink nucleotides for sequencing applications
AssigneePacific Biosciences of California, Inc.
InventorJonas Korlach
Life of this patent
Estimating the term…
Abstract
The present invention provides labeled phospholink nucleotides that can be used in place of naturally occurring nucleotide triphosphates or other analogs in template directed nucleic acid synthesis reactions and other nucleic acid reactions and various analyses based thereon, including DNA sequencing, single base identification, hybridization assays, and others.
Claims21
A composition, comprising a labeled phospholink nucleotide, wherein said labeled phospholink nucleotide comprises a structure that has formula:
- wherein
- B is a nucleobase;
- S is a sugar moiety;
- L is a bead comprising an optically detectable property;
- R1 is selected from oxygen and sulfur;
- R2 is a linker moiety;
- and
- n is an integer selected from 0 to 9.
A composition comprising a microfluidic flow cell, said composition further comprising:
- a. a nucleic acid polymerase immobilized on a surface of a substrate, wherein said substrate is contained within said microfluidic flow cell;
- and
- b. at least four differentially labeled nucleotides, wherein said labeled nucleotides comprise beads comprising a fluorophore.
A method of determining an identity of a nucleotide in a template nucleic acid sequence, said method comprising:
- a. providing said template nucleic acid sequence complexed with a polymerase enzyme capable of template dependent synthesis of a complementary nascent sequence as a first complex;
- b. contacting said first complex with a labeled phospholink nucleotide, wherein said labeled phospholink nucleotide comprises a bead comprising at least one fluorophore, and wherein said labeled phospholink nucleotide is complementary to a known nucleotide;
- and
- c. detecting whether said labeled phospholink nucleotide is incorporated into said nascent sequence, wherein incorporation of said labeled phospholink nucleotide is indicative that said complementary nucleotide is in a position in the template nucleic acid that is being processed by the polymerase enzyme.
A nucleic acid sequencing system, comprising:
- a body structure comprising a plurality of microfluidic channels;
- a source of one or more template nucleic acid, which template nucleic acid source is capable of being fluidly coupled to at least a first one of the microfluidic channels;
- a source of one or more sequencing reagent, which sequencing reagent source is capable of being fluidly coupled to the at least first microfluidic channel;
- wherein the source of one or more sequencing reagent comprises a set of at least four differently labeled nucleotide analogs, wherein said labeled nucleotide analogs comprise a fluorescent bead, and a fluid flow controller that flows the template nucleic acid and the one or more sequencing reagents into contact in the at least first microfluidic channel, whereby one or more sequencing product is formed, wherein a detector detects the one or more sequencing product, thereby determining at least a subsequence of the template nucleic acid.
Dependencies are inferred from the claim text.