US 12,319,962 B2
Methods for sequencing polynucleotides
Roberto Rigatti, Cambridge (GB); Niall Anthony Gormley, Cambridge (GB); and Jonathan Mark Boutell, Cambridge (GB)
Assigned to Illumina Cambridge Limited, Cambridge (GB)
Filed by Illumina Cambridge Limited, Cambridge (GB)
Filed on Mar. 8, 2022, as Appl. No. 17/689,237.
Application 17/689,237 is a continuation of application No. 16/436,525, filed on Jun. 10, 2019, granted, now 11,279,975, issued on Mar. 22, 2022.
Application 16/436,525 is a continuation of application No. 14/683,580, filed on Apr. 10, 2015, granted, now 10,329,613, issued on Jun. 25, 2019.
Application 14/683,580 is a continuation of application No. 13/219,422, filed on Aug. 26, 2011, granted, now 9,029,103, issued on May 12, 2015.
Claims priority of provisional application 61/377,732, filed on Aug. 27, 2010.
Prior Publication US 2022/0259651 A1, Aug. 18, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12P 19/34 (2006.01); C12Q 1/6874 (2018.01)
CPC C12Q 1/6874 (2013.01) 14 Claims
OG exemplary drawing
 
1. A method for determining sequences of polynucleotides that contain a first portion and a second portion from the same polynucleotide, comprising:
(a) providing a plurality of nucleic acid clusters attached to a surface, wherein the plurality of nucleic acid clusters is obtained by:
(i) providing a plurality of double-stranded polynucleotides attached to a surface, wherein a first portion on a first strand of each of the plurality of double-stranded polynucleotide molecules is attached to a first location of the surface and a second portion on a second strand of each of the plurality of double-stranded polynucleotide molecules is attached to a second location of the surface, wherein the first portion and the second portion are located at the opposite ends of two complementary strands of each of the plurality of double-stranded polynucleotide molecules, and wherein each of the plurality of double-stranded polynucleotide molecules has a known length and the first strand and the second strand are the two complementary strands of each of the plurality of double-stranded polynucleotide molecules;
(ii) separating the first portion on the first strand of each of the plurality of double-stranded polynucleotide molecules from the second portion on the second strand of each of the plurality of double-stranded polynucleotide molecules by denaturing the two complementary strands of each of the plurality of double-stranded polynucleotide molecules, thereby generating a plurality of denatured single polynucleotide strands such that the first portion on the first strand of each of the plurality of denatured single polynucleotide strands is immobilized on the first location of the surface and the second portion on the second strand of each of the plurality of denatured single polynucleotide strands is immobilized on the second location of the surface; and
(iii) amplifying the first portion on the first strand of each of the plurality of denatured single polynucleotide strands immobilized on the first location and amplifying the second portion on the second strand of each of the plurality of denatured single polynucleotide strands immobilized on the second location, thereby obtaining the plurality of nucleic acid clusters on the surface;
(b) identifying the locations of the plurality of nucleic acid clusters on the surface using an imaging device, and determining the distance from one cluster of the plurality of nucleic acid clusters to another cluster of the plurality of nucleic acid clusters on the surface;
(c) determining the sequences of the first portion and the second portion of the plurality of nucleic acid clusters; and
(d) comparing the location of each of the plurality of nucleic acid clusters on the surface, wherein the distance from one of the plurality of nucleic acid clusters to another of the plurality of nucleic acid clusters determines the probability of whether the sequence of the first portion of the one of the plurality of double-stranded polynucleotide molecules and the sequence of the second portion of the one of the plurality of double-stranded polynucleotide molecules are located on the same target polynucleotide molecule, and determining the sequences of the polynucleotides that contain the first portion and the second portion from the same polynucleotide.