Methods and systems for nucleic acid sequencing
Inventors
Lee, Linda • Menchen, Steven • Nikiforov, Theo • Almogy, Gilad • Oberstrass, Florian
Assignees
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Publication Number
US-12071653-B2
Publication Date
2024-08-27
Expiration Date
Abstract
The disclosure provides methods for sequencing nucleic acids using, including with nucleotide analogs and subsequently appended labels.
Core Innovation
The described invention determines a sequence of a template nucleic acid molecule by primer extension in which nucleotide analogs are contacted with a growing nucleic acid strand having sequence complementarity with the template nucleic acid molecule. The nucleotide analogs comprise a modified phosphate group comprising sulfur or selenium, so that the modified phosphate group is incorporated into the growing nucleic acid strand as the strand is extended.
After incorporation, at least one modified phosphate group of the nucleotide analogs is reacted with a labeling reagent comprising a label to generate a label-S-P, label-S-S-P, or label-Se-P group on the growing nucleic acid strand. One or more signals indicative of the label are detected to obtain sequencing information based on the labeled groups present on the growing nucleic acid strand.
A further conversion step converts the label-S-P, label-S-S-P, or label-Se-P group to an O−P group on the growing nucleic acid strand. The converting step provides one or more of a metal ion, an oxidant, an oxime, or a reducing agent to restore an O−P form substantially unreactive with the labeling reagent for continued sequencing cycles.
Claims Coverage
The independent claims are directed to a sequencing method that uses sulfur- or selenium-modified phosphate nucleotide analogs, labeling of the modified phosphate-derived groups for signal detection, and conversion back to an O−P group using selected converting reagents. The claims collectively present 6 inventive features, with specific oxidant and oxime selections used to further narrow some independent-claim formulations.
Incorporating sulfur or selenium modified phosphate nucleotide analogs
Contacting a nucleotide analog or two or more nucleotide analogs with a growing nucleic acid strand having sequence complementarity with a template nucleic acid molecule under conditions sufficient to incorporate the nucleotide analog into the growing nucleic acid strand, wherein the nucleotide analog comprises a modified phosphate group comprising sulfur or selenium.
Labeling modified phosphate-derived groups for detectable signals
Reacting the modified phosphate group of the nucleotide analog with a labeling reagent comprising a label to generate a label-S-P, label-S-S-P, or label-Se-P group on the growing nucleic acid strand.
Detecting signals indicative of the label
Detecting one or more signals indicative of the label.
Converting labeled sulfur/selenium phosphate groups to an O−P group
Converting the label-S-P, label-S-S-P, or label-Se-P group to an O−P group on the growing nucleic acid strand, including providing one or more of a metal ion, an oxidant, an oxime, or a reducing agent.
Limiting converting oxidants to specified oxidant classes
The converting step includes an oxidant selected from iodine, iodosobenzoate, and potassium peroxymonosulfate (oxone).
Limiting converting oximes to specified oxime identities
The converting step includes an oxime selected from 2-pyridine aldoxime, 4-pyridine aldoxime, obidoxime, HI-6, HLö-7, E-2-nitrobenzaldoxime, and E-4-nitrobenzldoxime.
The claims cover sequencing by detecting labels produced from sulfur- or selenium-modified phosphate nucleotide analogs incorporated into a growing complementary strand, then converting the labeled groups back to an O−P group using selected converting reagent classes, with some independent-claim formulations further limited by specified oxidants and oximes.
Stated Advantages
Documented Applications
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