METHODS AND COMPOSITIONS FOR SEQUENCING COMPLEMENTARY POLYNUCLEOTIDES
Disclosed herein, inter alia, are substrates, kits, and efficient methods of preparing and sequencing two or more regions of a double-stranded polynucleotide.
1 . A method of sequencing, comprising:
hybridizing a template polynucleotide to a first primer attached to a solid support and extending the first primer with a polymerase to generate a first strand;
contacting the first strand with a denaturant, thereby separating the template polynucleotide and the first strand;
removing the denaturant and hybridizing the first strand to a second primer attached to the solid support and extending the second primer with the polymerase to generate a second strand;
hybridizing an invasion primer to the second strand and extending the invasion primer with a polymerase, thereby generating an invasion strand, wherein the invasion primer is not covalently attached to the solid support; and
sequencing the first strand.
2 . The method of claim 1 , further comprising removing the first strand, removing the invasion strand, or both removing the first strand and removing the invasion strand.
3 . The method of claim 1 , further comprising removing the invasion strand and hybridizing a second invasion primer to the first strand and extending the second invasion primer with a polymerase, thereby generating a second invasion strand, wherein the second invasion primer is not covalently attached to the solid support.
4 - 6 . (canceled)
7 . The method of claim 1 , further comprising nicking the invasion strand at a cleavable site to generate a 3′ end and incorporating one or more nucleotides into the 3′ end of the nicked invasion strand with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand.
8 . The method of claim 2 , wherein removing the invasion strand comprises digesting the invasion strand using an exonuclease enzyme.
9 . The method of claim 1 , wherein the first primer is covalently attached to the solid support via a first linker and the second primer is covalently attached to the solid support via a second linker.
10 . (canceled)
11 . (canceled)
12 . The method of claim 1 , wherein the invasion primer comprises locked nucleic acids (LNAs), Bis-locked nucleic acids (bisLNAs), twisted intercalating nucleic acids (TINAs), bridged nucleic acids (BNAs), 2′-O-methyl RNA:DNA chimeric nucleic acids, minor groove binder (MGB) nucleic acids, morpholino nucleic acids, C5-modified pyrimidine nucleic acids, peptide nucleic acids (PNAs), phosphorothioate nucleic acids, or combinations thereof.
13 . The method of claim 1 , wherein the invasion primer comprises locked nucleic acids (LNAs), 2-amino-deoxyadenosine (2-amino-dA), trimethoxystilbene-functionalized oligonucleotides (TFOs), Pyrene-functionalized oligonucleotides (PFOs), peptide nucleic acids (PNAs), or aminoethyl-phenoxazine-dC (AP-dC) nucleic acids.
14 . The method of claim 1 , wherein the invasion primer is about 15 to about 35 nucleotides in length.
15 . The method of claim 1 , wherein the invasion primer comprises one or more locked nucleic acids (LNAs) at a 3′ end.
16 . The method of claim 1 , further comprising contacting the invasion primer with a recombinase, a crowding agent, a loading factor, a single-stranded binding (SSB) protein, or a combination thereof.
17 . The method of claim 1 further comprising contacting the second strand with one or more invasion-reaction mixtures.
18 . The method of claim 17 , wherein the invasion-reaction mixture comprises a plurality of invasion primers and a plurality of deoxyribonucleotide triphosphate (dNTPs).
19 . The method of claim 17 , wherein the invasion-reaction mixture comprises a denaturant, single-stranded DNA binding protein (SSB), or both a denaturant and single-stranded DNA binding protein (SSB).
20 . The method of claim 1 , wherein generating the invasion strand comprises a first plurality of invasion-primer extension cycles followed by a second plurality of invasion-primer extension cycles, wherein the reaction conditions for the first plurality of invasion-primer extension cycles are different.
21 . The method of claim 20 , wherein the reaction conditions for the first plurality of invasion-primer extension cycles comprise higher stringency hybridization conditions relative to the second plurality of invasion-primer extension cycles.
22 . The method of claim 1 , wherein generating the invasion strand comprises contacting the polynucleotide with a buffered solution comprising dimethyl sulfoxide (DMSO), betaine, or a combination of dimethyl sulfoxide (DMSO) and betaine.
23 . (canceled)
24 . The method of claim 1 , wherein sequencing comprises sequencing by synthesis, sequencing by binding, sequencing by ligation, or pyrosequencing.
25 .- 29 . (canceled)
30 . The method of claim 1 , wherein sequencing comprises:
hybridizing a sequencing primer to the first strand; incorporating one or more nucleotides into the sequencing primer with a polymerase to create an extension strand; and detecting the one or more incorporated nucleotides so as to identify each incorporated nucleotide in said extension strand.
31 . The method of claim 1 , wherein the invasion primer comprises one or more deoxyuracil nucleobases (dU).