Enzyme- and amplification-free sequencing

Patent metadata

Field	Value
Publication number	US-2016194701-A1
Application number	US-201514946386-A
Country	US
Kind code	A1
Filing date	Nov 19, 2015
Priority date	Nov 21, 2014
Publication date	Jul 7, 2016
Grant date	—

Abstract

The present invention relates to sequencing probes, methods, kits, and apparatuses that provide enzyme-free, amplification-free, and library-free nucleic acid sequencing that has long-read-lengths and with low error rate.

First claim

1 - 81 . (canceled)

82 . A method for sequencing a nucleic acid comprising steps of:
(1) hybridizing at least one sequencing probe to a target nucleic acid that is immobilized to a substrate; wherein said sequencing probe comprises: a target binding domain and a barcode domain;
wherein said target binding domain comprises at least four nucleotides and is capable of binding the immobilized target nucleic acid;
wherein said barcode domain comprises a synthetic backbone, said barcode domain comprising at least a first attachment region, said first attachment region comprising a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule and wherein said nucleic acid sequence of said first attachment region determines the position and identity of a first nucleotide in said immobilized target nucleic acid that is bound by a first nucleotide of said target binding domain and
(2) binding to the first attachment region a first complementary nucleic acid molecule comprising a detectable label or a first complementary nucleic acid molecule of a first reporter complex comprising a detectable label; (3) detecting the detectable label of the bound first complementary nucleic acid molecule or the detectable label of the bound first complementary nucleic acid molecule of the first reporter complex; (4) identifying the position and identity of the first nucleotide in the immobilized target nucleic acid; (5) contacting the first attachment region with a first hybridizing nucleic acid molecule lacking a detectable label thereby unbinding the first complementary nucleic acid molecule and binding to the first attachment region the first hybridizing nucleic acid molecule lacking a detectable label; (6) binding to a second attachment region a second complementary nucleic acid molecule comprising a detectable label or a second complementary nucleic acid molecule of a second reporter complex comprising a detectable label, said second attachment region comprising a nucleic acid sequence that determines the position and identity of a second nucleotide in the immobilized target nucleic acid that is bound by a second nucleotide of the target binding domain; (7) detecting the detectable label of the bound second complementary nucleic acid molecule or the detectable label of the bound second complementary nucleic acid molecule of the second reporter complex; and (8) identifying the position and identity of the second nucleotide in the immobilized target nucleic acid.

83 . (canceled)

84 . The method of claim 82 , wherein steps (5) and (6) occur sequentially or concurrently.

85 . The method of claim 82 , wherein steps (5) to (8) are repeated until each attachment region in the barcode domain has been sequentially bound by a complementary nucleic acid molecule comprising a detectable label or a complementary nucleic acid molecule of a reporter complex comprising a detectable label, and the detectable label of the sequentially bound complementary nucleic acid molecule or the detectable label of the sequentially bound complementary nucleic acid molecule of a reporter complex has been detected,
thereby identifying the linear order of nucleotides for a region of the immobilized target nucleic acid that was hybridized by the target binding domain of the sequencing probe.

86 . The method of claim 82 , wherein the target nucleic acid is first immobilized to a substrate by at least binding a first position of the target nucleic acid with a first capture probe that comprises a first affinity tag that selectively binds to a substrate.

87 . The method of claim 86 , wherein the target nucleic acid is elongated by applying a force sufficient to extend the target nucleic acid that is immobilized to a substrate at a first position.

88 . The method of claim 87 , wherein the force is gravity, hydrodynamic force, electromagnetic force, flow-stretching, a receding meniscus technique, or combinations thereof.

89 . The method of claim 88 , wherein the target nucleic acid is further immobilized to a substrate by binding an at least second position of the target nucleic acid with an at least second capture probe that comprises an affinity tag that selectively binds to the substrate.

90 . (canceled)

91 . The method of claim 89 , wherein the force can be removed once the second position of the target nucleic acid is immobilized to the substrate.

92 . (canceled)

93 . The method of claim 91 , wherein said immobilized target nucleic acid is elongated.

94 . The method of claim 82 , wherein said synthetic backbone comprises a polysaccharide, a polynucleotide, a peptide, a peptide nucleic acid, or a polypeptide.

95 . The method of claim 94 , wherein said synthetic backbone comprises single stranded-stranded DNA or single-stranded RNA or single-stranded PNA.

96 . The method of claim 95 , wherein said sequencing probe comprises a double-stranded DNA spacer between the target binding domain and the barcode domain.

97 . The method of claim 82 , wherein said first attachment region is adjacent to at least one flanking single-stranded polynucleotide.

98 . The method of claim 82 , wherein the first complementary nucleic acid is RNA, DNA or PNA or other polynucleotide analogue.

99 . The method of claim 82 , wherein the first nucleotide in said target binding domain is a modified nucleotide or a nucleic acid analogue.

100 . The method of claim 82 , wherein said barcode domain comprises at least a second attachment region, said second attachment region comprising a nucleic acid sequence capable of being bound by a second complementary nucleic acid molecule and wherein said nucleic acid sequence of said second attachment region determines the position and identity of a second nucleotide in said immobilized target nucleic acid that is bound by a second nucleotide of said target binding domain and
wherein the first complementary nucleic acid molecule is different from the second complementary nucleic acid molecule.

101 - 103 . (canceled)

104 . The method of claim 82 , wherein the first complementary nucleic acid molecule and the first hybridizing nucleic acid molecule lacking a detectable label comprise the same nucleic acid sequence.

105 . The method of claim 97 , wherein the first hybridizing nucleic acid molecule lacking a detectable label comprises a nucleic acid sequence complementary to a flanking single-stranded polynucleotide adjacent to said first attachment region.

106 - 113 . (canceled)

114 . The method of claim 82 , wherein the number of nucleotides in a target binding domain equals the number of attachment regions in the barcode domain.

115 . The method of claim 82 , wherein the number of nucleotides in a target binding domain is at least one more than the number of attachment regions in the barcode domain.

116 . The method of claim 82 , wherein at least the first attachment region branches from the synthetic backbone.

117 - 123 . (canceled)

124 . The method of claim 82 , wherein at least one position in a barcode domain has a greater number of attachment regions as another position.

125 - 127 . (canceled)

128 . The method of claim 82 , wherein each reporter complex comprising a detectable label comprises a complementary nucleic acid molecule directly linked to a primary nucleic ac

Assignees

• Nanostring Technologies Inc

Inventors

• Beechem Joseph M
• Khafizov Rustem

Classifications

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2537/149

  Sequential reactions · CPC title

• C12Q2525/113

  incorporating modified backbone · CPC title