Dna sequencing using controlled strand displacement

Patent metadata

Field	Value
Publication number	US-2016237488-A1
Application number	US-201615040906-A
Country	US
Kind code	A1
Filing date	Feb 10, 2016
Priority date	Feb 17, 2015
Publication date	Aug 18, 2016
Grant date	—

Abstract

This application discloses methods of producing a DNA strand for sequencing, as well as genetic constructs, libraries, and arrays using DNA strands produced according to these methods. The application also discloses methods of sequencing using the DNA strands, genetic constructs, libraries, and arrays produced. In certain aspects, DNA being sequenced includes a target sequence and at least one adaptor sequence.

First claim

1 . A method of producing a DNA strand for sequencing, comprising
a) providing a template DNA polynucleotide comprising a first target DNA sequence interposed between a first adaptor 3′ to the first target DNA sequence and a second adaptor 5′ to the first target DNA sequence, and optionally comprising a third adaptor 3′ to the first adaptor and a second target DNA sequence interposed between the first adaptor and the third adaptor, wherein the template DNA polynucleotide is immobilized on a substrate, b) combining a first primer with the immobilized template DNA polynucleotide, and hybridizing the first primer to a first primer binding sequence in the first adaptor, wherein the first primer is not immobilized on the substrate when it is combined with the immobilized template DNA polynucleotide; c) extending the first primer using a first DNA polymerase to generate a second strand, wherein the second strand comprises a sequence complementary to the first target DNA sequence and a sequence complementary to at least part of the second adaptor; d) combining a second primer with the immobilized template DNA polynucleotide, hybridizing a second primer to a second primer binding sequence, wherein the second primer binding sequence is 3′ to the first primer binding sequence, wherein the second primer is not immobilized on the substrate when it is combined with the immobilized template DNA polynucleotide; and e) extending the second primer using a DNA polymerase having strand-displacement activity to generate a third strand, wherein extending the second primer to generate the third strand partially displaces the second strand, thereby producing a partially hybridized second strand having:
(i) a hybridized portion that is hybridized to the template DNA polynucleotide, and
(ii) an unhybridized overhang portion that contains a sequence that is complementary to the first target DNA sequence and a sequence that is complementary to at least part of the second adaptor, wherein the unhybridized portion is 5′ in the second strand to the hybridized portion

2 . The method of claim 1 , further comprising:
f) hybridizing a sequencing oligonucleotide to the sequence in the third strand that is complementary to at least part of the second adaptor, and g) determining at least part of the sequence that is complementary to the first target DNA sequence.

3 . The method of claim 1 , wherein the first adaptor, the second adaptor, and the third adaptor if present, have the same nucleotide sequence.

4 . The method of claim 1 , wherein said first DNA polymerase and said DNA polymerase having strand-displacement activity are the same polymerase.

5 . The method of claim 1 , wherein the second primer binding sequence, to which the second primer is hybridized, is in the first adaptor.

6 . The method of claim 1 , wherein the template DNA polynucleotide comprises the third adaptor and the second primer binding sequence is in the third adaptor.

7 . The method of claim 1 , wherein the template DNA polynucleotide comprises a DNA concatemer, and the first target DNA sequence and the second target DNA sequence have the same nucleotide sequence.

8 . The method of claim 4 , wherein the first primer and the second primer are hybridized or extended in the same reaction.

9 . The method of claim 1 , wherein the template DNA polynucleotide comprises a DNA concatemer and the first primer and the second primer have the same nucleotide sequence.

10 . The method of claim 1 , wherein in step d) hybridizing a plurality of second primers to a plurality of second primer binding sequences, and wherein the plurality of second primers comprise extendable and non-extendable primers.

11 . The method of claim 1 , wherein extension of the second primer in terminated at a fixed time interval of 5 min, 10 min, 20 min, 30 min, 40 min or 60 min, and wherein extension is terminated by chemical termination and/or addition of ddNTPs.

12 . The method of claim 1 , wherein the extending the second primer to generate the third strand is controlled by temperature, enzyme concentration, and primer concentration.

13 . The method of claim 1 , wherein each of the template DNA is deposited on arrays, beads, wells, or droplets.

14 . The method of claim 1 , wherein the sequencing is sequencing by synthesis, pyrosequencing, or sequencing by ligation.

15 . An array of DNA complexes, wherein the array is a support comprising an array of discrete areas, wherein a plurality of the areas comprise
(a) single-stranded DNA concatemers, each concatemer comprising a plurality of monomers, each monomer comprising a target sequence and an adaptor sequence; (b) wherein each of a plurality of monomers of at least a subset of the DNA concatemers in (a) comprise,
(i) partially hybridized thereto, a second DNA strand, where each second strand DNA comprises a portion complementary to the target sequence and a portion complementary to at least part of the adaptor sequence, and wherein a portion of the second strand is not hybridized to the concatemer and a portion of the second strand complementary to at least part of the adaptor is hybridized to the adaptor, and
(ii) a third DNA strand comprising a portion complementary to, and hybridized to, the target sequence; and
(c) wherein each of at least a subset of the plurality of monomers of (b) comprises a fourth DNA strand hybridized to the third DNA strand at a hybridization site, wherein the fourth DNA strand comprises at least a portion of the sequence of the adaptor and the hybridization site is complementary to at least part of the second adaptor sequence.

16 . An array of DNA complexes, wherein the array is a support comprising an array of discrete areas, wherein a plurality of the areas comprise
(a) a clonal cluster of double or single-stranded DNAs, each DNA comprising a target sequence flanked by a first adaptor and a second adaptor; (b) wherein each of a plurality of DNAs of at least a subset of the clusters in (a) comprise, (i) partially hybridized thereto, a second DNA strand, where each second strand DNA comprises a portion complementary to the target sequence and a portion complementary to at least part of first adaptor sequence, and wherein a portion of the second strand complementary to the target sequence is not hybridized to the DNA and a portion of the second strand complementary to at least part of the first adaptor is hybridized to the DNA, and (ii) a third DNA strand comprising a portion complementary to, and hybridized to, the target sequence and a portion complementary to, and hybridized to, the second adaptor sequence; and (c) wherein each of at least a subset of the plurality of DNAs of (b) comprises a fourth DNA strand hybridized to the third DNA strand at a hybridization site, wherein the fourth DNA strand comprises at least a portion of the sequence of the second adaptor and the hybridization site is complementary to at least part of the second adaptor sequence.

17 . A composition or system comprising an array according to claim 15 and an enzyme selected from the group consisting of a DNA ligase and a DNA polymerase, wherein the DNA polymerase has strand displacement activity.

18 . A composition or system comprising an array according to claim 16 and an enzyme selected from the group consisting of a DNA ligase and a DNA polymerase, wherein the DNA polymerase has strand displacement activity.

Assignees

• Complete Genomics Inc

Inventors

• Ke Rongqin
• Drmanac Snezana
• Drmanac Radoje
• Cai Guangyang
• Callow Matthew

Classifications

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q1/6874 Primary

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