Sequencing of modified nucleic acid molecules
Modified nucleic acids are used for many purposes in research, diagnostic, and treatment protocols. However, direct sequencing of such molecules is generally considered to require special conditions. Methods of making a cDNA using a modified nucleic acid molecule and sequencing methods of sequencing a modified nucleic acid molecule are described.
1 . A method for determining the nucleotide sequence of a modified nucleic acid including 2′-modified nucleotides, comprising:
(a) obtaining a sample of a modified nucleic acid comprising a multiplicity of 2′-modified nucleotides,
(b) synthesizing a first cDNA complementary to said modified nucleic acid using a reverse transcriptase;
(c) synthesizing a second cDNA complementary to said first cDNA using a DNA polymerase to form a double-stranded cDNA;
(d) producing multiple double-stranded copies of said double-stranded cDNA; and
(e) sequencing said double-stranded copies;
wherein said modified nucleic acid includes a multiplicity of 2′-fluorinated nucleotides and a multiplicity of 2′-O-methylated nucleotides.
2 . The method of claim 1 , wherein said 2′-fluorinated nucleotides comprise between 10% and 70% of the total nucleotides in said modified nucleic acid.
3 . The method of claim 1 , wherein said 2′-fluorinated nucleotides comprise at least 40% of the total nucleotides in said modified nucleic acid.
4 . The method of claim 1 , wherein said 2′-O-methylated nucleotides comprise between 10% and 70% of the total nucleotides in said modified nucleic acid.
5 . The method of claim 1 , wherein said 2′-O-methylated nucleotides comprise at least 40% of the total nucleotides in said modified nucleic acid.
6 . The method of claim 1 , wherein said 2′-fluorinated nucleotides comprise between 10% and 70% of the total nucleotides in said modified nucleic acid and said 2′-O-methylated nucleotides comprise between 10% and 70% of the total nucleotides in said modified nucleic acid.
7 . The method of any one of claims 1 - 6 , wherein said modified nucleic acid further comprises a 5′ covalent modification comprising a large hydrophilic moiety.
8 . A method for determining the nucleotide sequence of a modified nucleic acid including a 5′ or 3′ large hydrophilic moiety, comprising:
(a) obtaining a sample of a modified nucleic acid comprising a 5′ or 3′ large hydrophilic moiety,
(b) synthesizing a first cDNA complementary to said modified nucleic acid using a reverse transcriptase;
(c) synthesizing a second cDNA complementary to said first cDNA using a DNA polymerase to form a double-stranded cDNA;
(d) producing multiple double-stranded copies of said double-stranded cDNA; and
(e) sequencing said double-stranded copies.
9 . The method of claim 8 , wherein said large hydrophilic moiety is selected from the group consisting of branched or straight-chain, substituted or unsubstituted, homopolymers or heteropolymers of alkyl, akenyl, aryl, or heterocyclic groups
10 . The method of claim 9 , wherein said large hydrophilic moiety is a polyethylene glycol moiety.
11 . The method of claim 9 , wherein said large hydrophilic moiety is a 10-50 kDa polyethylene glycol moiety.
12 . The method of any one of claims 8 - 11 , wherein said modified nucleic acid further comprises at least one 2′-fluorinated nucleotide or at least one 2′-O-methylated nucleotide.
13 . The method of claim 1 , further comprising:
(i) purifying said first cDNA after step (b); and
(ii) polyadenylating the 3′-end of said first cDNA prior to step (c).
14 . The method of claim 1 , wherein step (d) comprises:
(i) ligating said double-stranded cDNA into a cloning vector;
(ii) transforming host cells with said cloning vector;
(iii) isolating said cloning vector from descendants of said host cells;
(iv) isolating said double-stranded copies from said host cells.
15 . The method of claim 1 wherein step (d) comprises performing the polymerase chain reaction using said double-stranded cDNA as an original template molecule.
16 . A method of synthesizing a DNA complementary to a modified nucleic acid, the modified nucleic acid comprising 2′-modified nucleotides, the method comprising:
(a) obtaining a sample of a modified nucleic acid comprising a multiplicity of 2′-modified nucleotides,
(b) synthesizing a first cDNA complementary to said modified nucleic acid using a reverse transcriptase,
wherein said modified nucleic acid includes a multiplicity of 2′-fluorinated nucleotides and a multiplicity of 2′-O-methylated nucleotides.
17 . The method of claim 16 , wherein said modified nucleic acid further comprises a 5′ covalent modification comprising a large hydrophilic moiety.
18 . The method of claim 16 , wherein said large hydrophilic moiety is selected from the group consisting of branched or straight-chain, substituted or unsubstituted, homopolymers or heteropolymers of alkyl, akenyl, aryl, or heterocyclic groups.
19 . The method of claim 18 , wherein said large hydrophilic moiety is a polyethylene glycol moiety.
20 . The method of claim 18 , wherein said large hydrophilic moiety is a 10-50 kDa polyethylene glycol moiety.
21 . The method of claim 16 , wherein the modified nucleic acid further comprises a modified 3′ terminus.