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.
111 Citations
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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Current AssigneePacific Bioscience of California Incorporated (L'Oreal SA)
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Original AssigneePacific Bioscience of California Incorporated (L'Oreal SA)
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InventorsTravers, Kevin, Otto, Geoff, Turner, Stephen, Heiner, Cheryl, Ma, Congcong
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Primary Examiner(s)Lu, Frank W
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Application NumberUS13/019,220Publication NumberTime in Patent Office651 DaysField of Search435/6.1, 435/6.12, 435/91.1, 435/91.2, 435/91.51, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3US Class Current435/91.2CPC Class CodesC12Q 1/6806 Preparing nucleic acids for...C12Q 1/6855 Ligating adaptorsC12Q 1/6869 Methods for sequencingC12Q 1/689 for bacteriaC12Q 2525/155 incorporating/generating a ...C12Q 2525/301 Hairpin oligonucleotidesC12Q 2525/307 Circular oligonucleotidesC12Q 2531/125 Rolling circleC12Q 2535/122 Massive parallel sequencingG16B 30/00 ICT specially adapted for s...G16B 30/10 Sequence alignment; Homolog...
