Reversible di-nucleotide terminator sequencing
AssigneeApplied Biosystems, LLC
InventorGerald ZON
Life of this patent
Estimating the term…
Abstract
The present teachings provide methods, compositions, and kits for synthesizing and sequencing nucleic acids. In some embodiments, reversible di-nucleotide compounds are employed along with cleaving reactions that remove a label and a blocking moiety. Improved sequencing efficiency is achieved by the rapid polymerase-mediated incorporation of reversible di-nucleotide compounds. In some embodiments, the di-nucleotides do not contain conventional nucleotide triphosphates, but rather employ amino acid phosphoramidate nucleotides (AAPNs).
Claims21
A method for determining a sequence of nucleotides in a template, the method comprising:
- (a) hybridizing a primer to a template;
- (b) extending the primer in the presence of a reversible di-nucleotide terminator, wherein the reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide;
- (c) identifying a family of the reversible di-nucleotide terminator;
- (d) generating an extendable terminus on the incorporated reversible di-nucleotide terminator;
- (e) repeating steps (b), (c), and (d) a suitable number of times to form an extension product hybridized to the template;
- (f) removing the extension product;
- (g) hybridizing a shifted-primer to the template;
- (h) extending the primer in the presence of a reversible di-nucleotide terminator, wherein the reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide;
- (i) identifying a family of the reversible di-nucleotide terminator;
- j) generating an extendable terminus on the incorporated reversible di-nucleotide terminator;
- (k) repeating steps (h), (i), and (j) a suitable number of times;
- and,
- (l) sequencing the template.
A method for determining information about a sequence of nucleotides in a template using a first collection of at least 2 distinguishably labeled reversible di-nucleotide terminators, the method comprising;
- (a) extending a primer along the template by incorporating a reversible di-nucleotide terminator to form an extension product, wherein the reversible di-nucleotide terminator is a member of the collection of distinguishably labeled reversible di-nucleotide terminator;
- (b) detecting a label associated with the reversible di-nucleotide terminator;
- (c) repeating steps (a) and (b) until an ordered list of reversible di-nucleotide terminator family names is obtained;
- and,
- (d) using the ordered list of reversible di-nucleotide terminator family names to eliminate one or more possibilities for the sequence of nucleotides of the template.
A collection of distinguishably labeled reversible di-nucleotide terminator families, wherein each family comprises a collection of di-nucleotides, and wherein the collection of di-nucleotides in a probe family comprise the same label.
A method for determining a sequence of nucleotides in a template, the method comprising:
- (a) hybridizing a primer to a template;
- (b) extending the primer in the presence of a reversible di-nucleotide terminator, wherein the reversible di-nucleotide terminator comprises a first nucleotide and a second nucleotide, to incorporated the reversible di-nucleotide terminator;
- (c) identifying two bases in the template based on a dye associated with the incorporated di-nucleotide terminator;
- (d) generating an extendable terminus on the incorporated reversible di-nucleotide terminator,
- (e) repeating steps (b), (c), and (d) a suitable number of times to determine the sequence of nucleotides in the template.
A kit for determining information about a sequence of nucleotides in a template polynucleotide comprising;
- a collection of distinguishably labeled reversible di-nucleotide terminator families, wherein each family comprises a collection of reversible di-nucleotide terminators, and wherein the collection of di-nucleotides in a family comprise the same label;
- and,
- a suitable polymerase.
Dependencies are inferred from the claim text.