Methods for flip-strand immobilizing and sequencing nucleic acids

A method for immobilizing a polynucleotide, comprising:

• a) attaching a solid surface to a single-stranded oligonucleotide which is joined to a template polynucleotide, (i) wherein the single-stranded oligonucleotide includes a first priming sequence (P1) and includes a nucleic acid base or linkage that is susceptible or resistant to enzymatic cleavage and forms a secondary structure that is a hairpin or U-shaped secondary structure, and (ii) wherein the template polynucleotide includes a P2 priming sequence;
• b) hybridizing a P2′ primer to the P2 priming sequence;
• c) extending the P2′ primer with a primer extension reaction to generate a complement polynucleotide;
• d) joining the complement polynucleotide to the single-stranded oligonucleotide thereby immobilizing the complement polynucleotide to the solid surface;
• and
• e) conducting an enzymatic reaction on the susceptible or resistant enzyme cleavage site to remove the template polynucleotide from the solid surface so as to generate an immobilized complement polynucleotide.

A method for immobilizing a polynucleotide, comprising:

• a) attaching a solid surface to a single-stranded oligonucleotide which is joined to a template polynucleotide, (i) wherein the single-stranded oligonucleotide includes a first priming sequence (P1) and includes an enzyme-cleavable base (CS) and includes a nucleotide sequence that mediates triple-strand formation (XS), and (ii) wherein the template polynucleotide includes a P2 priming sequence;
• b) hybridizing a P2′ primer to the P2 priming sequence;
• c) conducting a primer extension reaction on the P2′ primer to generate a complement polynucleotide;
• d) reacting the nucleotide sequence that mediates triple-strand formation (XS) and the complement polynucleotide with a triplex-forming oligonucleotide (XO) under suitable conditions so as to form a triple strand;
• e) cleaving the enzyme-cleavable base (CS) with an enzyme to remove the template polynucleotide from the solid surface so as to generate an immobilized complement polynucleotide.

A method for immobilizing a polynucleotide, comprising:

• a) attaching a solid surface to a first and second single-stranded oligonucleotide, wherein the first single-stranded oligonucleotide is joined to a template polynucleotide, (i) wherein the single-stranded oligonucleotide includes a first priming sequence (P1) and includes a first enzyme-cleavable site and (ii) wherein the template polynucleotide includes a second priming sequence (P2);
• and wherein the second single-stranded oligonucleotide is joined to an aminated oligonucleotide, wherein the second single-stranded oligonucleotide includes a second enzyme-cleavable base;
• b) hybridizing a P1/P2′ hybrid primer to the P2 priming sequence;
• c) extending the second priming sequence (P2) so as to generate an extended template polynucleotide having a first priming sequence (P1), first enzyme-cleavable base, a template polynucleotide sequence, a second priming sequence (P2), and an extended P1′ sequence;
• d) folding the extended template polynucleotide on itself, so as to hybridize the P1 sequence with the P1′ sequence;
• e) removing the first enzyme-cleavable base with an enzymatic reaction so as to leave the first single-stranded oligonucleotide having the first primer sequence (P1) immobilized to the bead, and so as to leave the extended template polynucleotide hybridized to the first primer sequence (P1) that is immobilized to the bead.