Self-assembled single molecule arrays and uses thereof
The present invention provides methods of making and using self-assembled arrays of single polynucleotide molecules for carrying out a variety of large-scale genetic measurements, such as gene expression analysis, gene copy number assessment, and the like. Random arrays used in the invention are “self-assembled” in the sense that they are formed by deposition of polynucleotide molecules onto a surface where they become fixed at random locations. The polynucleotide molecules fixed on the surface are then identified by direct sequence determination of component nucleic acids, such as incorporated probe sequences, or by other decoding schemes. Such identification converts a random array of determinable polynucleotides, and their respective probes into an addressable array of probe sequences.
1. A method of determining sequence information for a target polynucleotide, comprising:
(a) providing a plurality of concatemers, each concatemer having been obtained by:
(i) forming a first circular DNA comprising a fragment of said target polynucleotide and a first adaptor, wherein the first adaptor comprises a binding site for a restriction enzyme that cleaves DNA at a cleavage site separated from said binding site by at least six nucleotides;
(ii) forming a linearized DNA by a process that comprises cleaving the first circular DNA at a site that is internal to the target polynucleotide using a restriction endonuclease that binds to said binding site, whereby the linearized DNA comprises the first adaptor flanked on each side by a portion of said fragment;
(iii) forming a second circular DNA comprising said linearized DNA and a second adaptor;
(iv) amplifying the second circular DNA to form said concatemer;
(b) forming an array of said concatemers bound to a surface; and
(c) determining target sequences both upstream and downstream from the second adaptor by a process that comprises hybridizing oligonucleotides to the second adaptor in a plurality of the concatemers in the array.
2. The method of claim 1 , wherein step (c) comprises:
(i) hybridizing an anchor probe to a probe hybridization site in said second adaptor;
(ii) hybridizing a sequencing probe to a target sequence of the target polynucleotide adjacent to the hybridized anchor probe; then
(iii) ligating the sequencing probe and the anchor probe; and
(iv) detecting the probe ligated in step (iii) to identify said nucleotide(s) in the target sequence.
3. The method of claim 2 , wherein steps (i) through (iv) of step (c) are repeated to identify a nucleotide sequence of the target polynucleotide.
4. The method of claim 1 comprising sequentially hybridizing anchor probes to hybridization sites on both ends of the second adaptor.
5. The method of claim 1 , wherein the target polynucleotide is genomic DNA.
6. The method of claim 1 , wherein the surface comprises more than 100,000 concatemers per square millimeter.
7. The method of claim 6 , wherein at least 70% of said concatemers are optically resolvable.
8. The method of claim 1 , wherein the concatemers are bound to discrete spaced-apart regions on the surface.
9. The method of claim 8 , wherein the concatemers are randomly disposed amongst said discrete spaced apart regions.
10. The method of claim 8 , wherein each discrete spaced apart region has an area of less than 1 μm 2 .
11. The method of claim 8 , wherein the concatemers are bound to the discrete spaced-apart regions non-covalently.
12. The method of claim 8 , wherein at least 80% of the discrete spaced apart regions have one concatemer attached.
13. The method of claim 1 , wherein step (ii) comprises cleaving the first circular DNA on two sites outside of the first adaptor.
14. The method of claim 1 , wherein step (c) comprises hybridizing oligonucleotides to the second adaptor at both ends of the adaptor so as to determine sequence of the target fragment beside or near both the first and the second adaptor in both directions.
15. The method of claim 1 , wherein step (c) comprises hybridizing anchor probes to the second adaptor at or near both ends of the adaptor, hybridizing labeled sequencing probes to the target polynucleotide at positions adjacent to the anchor probes, and ligating sequencing probes to adjacent anchor probes.