US 8,445,194 B2Granted

Single molecule arrays for genetic and chemical analysis

AssigneeCallida Genomics, Inc.

InventorsRadoje Drmanac, Matthew J. Callow, Snezana Drmanac, Brian K. Hauser, George Yeung

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Abstract

Random arrays of single molecules are provided for carrying out large scale analyses, particularly of biomolecules, such as genomic DNA, cDNAs, proteins, and the like. In one aspect, arrays of the invention comprise concatemers of DNA fragments that are randomly disposed on a regular array of discrete spaced apart regions, such that substantially all such regions contain no more than a single concatemer. Preferably, such regions have areas substantially less than 1 μm2 and have nearest neighbor distances that permit optical resolution of on the order of 109 single molecules per cm2. Many analytical chemistries can be applied to random arrays of the invention, including sequencing by hybridization chemistries, sequencing by synthesis chemistries, SNP detection chemistries, and the like, to greatly expand the scale and potential applications of such techniques.

Claims20

Dependent claims

A method of identifying a sequence of a target polynucleotide, said method comprising:

a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of said discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and
wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises:
i) a first target sequence of said target polynucleotide;
ii) an adaptor comprising a Type IIs endonuclease restriction site, wherein said first target sequence is adjacent to said adaptor;
b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor;
c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence;
d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex;
e) detecting said ligated complex thereby identifying a nucleotide of said target polynucleotide;
f) stripping off said ligated complex;
g) repeating steps (b) through (f) in order to determine said sequence of said target polynucleotide.

7 more elements

A method of identifying a sequence of a target polynucleotide, said method comprising:

a) providing a substrate comprising a plurality of discrete regions, wherein a plurality of concatemers are randomly disposed on said plurality of discrete regions, wherein at least a majority of said discrete regions comprises a single concatemer disposed thereon, wherein at least a majority of said concatemers at said plurality of discrete regions are optically resolvable, and
wherein each concatemer comprises a plurality of monomeric units and each monomeric unit comprises:
i) a first target sequence of said target polynucleotide;
ii) an adaptor, wherein said first target sequence is adjacent to said adaptor;
b) applying a first set of probes to said substrate such that one or more probes from said first set hybridizes to said adaptor;
c) applying a second set of probes to said substrate such that one or more probes from said second set hybridizes to said first target sequence;
d) ligating probes from said first set and probes from said second set that are hybridized to adjacent sequences of said monomeric unit to form a ligated complex;
e) detecting said ligated complex;
thereby identifying said sequence of said target polynucleotide.

6 more elements

Dependencies are inferred from the claim text.

Facts

Application: 11/451,691Filed 13 Jun 2006 · us provisional application
Priority: 15 Jun 2005
Published: US 2007/0099208 A13 May 2007
Granted: 21 May 2013
Term ends: Not estimatedThe source records don’t support an estimate.

Classification23 codes

C12Q 1/6874
Methods for sequencing: involving nucleic acid arrays, e.g. sequencing by hybridisation
C07H 21/04
Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of…: with…
C07K 1/047
On carriers: simultaneous synthesis of different peptide species; Peptide libraries
C12Q 1/6806
Involving nucleic acids: preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Family

Application60/690,771Us provisional application · filed 15 Jun 2005
Application60/725,116Us provisional application · filed 7 Oct 2005
Application60/776,415Us provisional application · filed 24 Feb 2006
US 2007/0099208 A1Pre-grant publication · published 3 May 2007
US 8,445,194 B2This patent

From the US filing history. Foreign family members aren’t listed.