Diagnostic sequencing with small nucleic acid circles
First Claim
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1. A method for sequencing a circular template nucleic acid comprising:
- carrying out template dependent nucleic acid synthesis in a reaction mixture comprising a polymerase, a primer, nucleotides or nucleotide analogs, and a circular template nucleic acid under conditions which allow for identification of added nucleotides or nucleotide analogs during the synthesis, and repeatedly sequencing the circular template nucleic acid, wherein the circular template nucleic acid comprises both a double-stranded segment and one or more linking oligonucleotides that comprise at least one primer recognition sequence, wherein the one or more linking oligonucleotides form a connecting loop between a 3′
end of one strand of the double stranded segment and a 5′
end of another strand of the double stranded segment, and the primer is complementary to the at least one primer recognition sequence.
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Abstract
Methods, substrates, and systems for diagnostic sequencing are provided. Small circles of nucleic acids from about 10 bases to about 200 bases can be sequenced, for example using template dependent sequencing by synthesis. The use of small circles of nucleic acids allows for repeated sequencing of the same portions of the nucleic acid, providing for higher accuracy sequence determinations.
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13 Claims
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1. A method for sequencing a circular template nucleic acid comprising:
carrying out template dependent nucleic acid synthesis in a reaction mixture comprising a polymerase, a primer, nucleotides or nucleotide analogs, and a circular template nucleic acid under conditions which allow for identification of added nucleotides or nucleotide analogs during the synthesis, and repeatedly sequencing the circular template nucleic acid, wherein the circular template nucleic acid comprises both a double-stranded segment and one or more linking oligonucleotides that comprise at least one primer recognition sequence, wherein the one or more linking oligonucleotides form a connecting loop between a 3′
end of one strand of the double stranded segment and a 5′
end of another strand of the double stranded segment, and the primer is complementary to the at least one primer recognition sequence.- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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