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US 12,351,865 B2Granted
Methods, compositions, systems, apparatuses and kits for nucleic acid amplification
AssigneeLife Technologies Corporation
InventorsBin Li, Kai Qin Lao, Jennifer O'Neil, Jennifer Kunkel, Kellie Haley, Rachel Kasinskas, Zhaochun Ma, Pius Brzoska
Life of this patent
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Abstract
Novel methods of generating a localized population of immobilized clonal amplicons on a support are provided.
Claims17
A method of primer extension in isothermal conditions, comprising:
- a) hybridizing a first primer molecule (“first forward primer”) to a complementary primer-binding sequence (“reverse-strand PBS”) on a nucleic acid strand (“reverse strand”);
- wherein the first forward primer is immobilized to a solid support;
- b) generating an extended first forward strand that is a full-length complement of the reverse strand and is hybridized thereto, by extending the first forward primer molecule using the reverse strand as template;
- c) causing or allowing the double-stranded reverse-strand PBS to denature or breathe;
- d) hybridizing a second primer molecule (“second forward primer”) to the reverse-strand PBS where the reverse strand is also hybridized to the first forward strand;
- wherein the second forward primer is immobilized to the solid support.
3 more elements
A composition comprising:
- a double-stranded nucleic acid comprising a reverse strand and a forward strand, the reverse strand comprising a primer-binding sequence (“reverse-strand PBS”), the forward strand not hybridized to the reverse-strand PBS, and a second primer molecule (“second forward primer”) hybridized to the reverse-strand PBS, wherein the second forward primer is immobilized to a solid support, wherein at least 60% of nucleotide bases of the second forward primer are adenine, thymine or uracil or are complementary to adenine, thymine or uracil.
Dependencies are inferred from the claim text.