Single molecule arrays for genetic and chemical analysis
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 μm 2 and have nearest neighbor distances that permit optical resolution of on the order of 10 9 single molecules per cm 2 . 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.
1. 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 majority of said plurality of discrete regions comprises a single concatemer comprising a plurality of monomeric units,
wherein each monomeric unit comprises a first target sequence of said target polynucleotide and an adaptor, and said first target sequence is adjacent to said adaptor,
and wherein said substrate is prepared by a photolithography method such that said plurality of discrete regions comprise a functional group for non-covalent attachment of said concatemer;
(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) detected said ligated complex, thereby identifying a nucleotide of said target polynucleotide;
(f) removing said ligated complex;
(g) repeating steps (b) through (f) to determine said sequence of said target polynucleotide.
2. The method of claim 1 , wherein said substrate is a planar material that comprises silica.
3. The method of claim 1 , wherein said functional group comprises an amine group.
4. The method of claim 3 , wherein said amine group is an amino group.
5. The method of claim 1 , wherein one or more probes from said first set or from said second set comprise a detectable label and wherein detecting said ligated complex comprises detecting said detectable label.
6. The method of claim 5 , wherein said detectable label comprises a fluorophore.
7. The method of claim 1 , wherein one or more probes from said first set comprise one or more degenerate nucleotides.
8. The method of claim 1 , wherein forming said ligated complex produces a detectable signal, and wherein said detecting step (e) comprises detecting said detectable signal.
9. The method of claim 1 , wherein said concatemers are formed by a method comprising:
(a) providing fragments of said target polynucleotide;
(b) ligating an adaptor to a terminus of a plurality of said fragments;
(c) circularizing said fragments ligated to said adaptors to form circular products;
(d) generating a concatemer from at least one of said circular products, thereby forming said concatemers.
10. The method of claim 9 , wherein said generating is through a rolling circle replication reaction.
11. The method of claim 9 , wherein said fragments substantially cover said target polynucleotide.
12. The method of claim 1 , wherein at least 70% of said plurality of discrete regions comprise a single concatemer.
13. The method of claim 1 , wherein said target polynucleotide is human genomic DNA.