Articles having localized molecules disposed thereon and methods of producing same
Methods of producing substrates having selected active chemical regions by employing elements of the substrates in assisting the localization of active chemical groups in desired regions of the substrate. The methods may include optical, chemical and/or mechanical processes for the deposition, removal, activation and/or deactivation of chemical groups in selected regions of the substrate to provide selective active regions of the substrate.
1. A method of identifying a sequence of a nucleic acid molecule, comprising:
providing nucleic acid polymerase/template/primer complexes within discrete observation regions on a substrate surface, and detecting sequential addition of nucleotides or nucleotide analogs in a template dependent manner to identify a sequence of incorporation of the nucleotides or nucleotide analogs in the observation regions;
wherein the nucleotides or nucleotide analogs comprise at least one nucleotide analog that is labeled on a phosphate group, addition of which nucleotide analog results in release of a labeled polyphosphate;
wherein the substrate comprises a cladding layer and a transparent layer; wherein the observation regions are disposed within zero mode waveguides that are disposed through the cladding layer to the transparent layer that forms the bottom of the zero mode waveguides; wherein either the polymerase or the template or the primer component of the nucleic acid polymerase/template/primer complexes is immobilized on the substrate surface; wherein density of the immobilized component on the bottom of the zero mode waveguides is 100 times or more greater than density of the immobilized component on the substrate surface other than the bottom of the zero mode waveguides; and wherein detecting sequential addition of nucleotides or nucleotide analogs in a template dependent manner comprises providing illumination to and receiving electromagnetic radiation from the observation regions through the transparent layer.
2. The method of claim 1 , wherein adjacent zero mode waveguides are in contact with a fluid and are separated by sufficient distance to substantially prevent one or more products or reactants from one of the observation regions from reaching the adjacent observation region.
3. The method of claim 1 , wherein the substrate surface between the zero mode waveguides comprises a printable material.
4. The method of claim 3 , wherein the printable material is present on the top surface of the substrate surface but does not penetrate into the zero mode waveguides.
5. The method of claim 1 , wherein the substrate surface between the zero mode waveguides comprises a hydrophilic or hydrophobic material.
6. The method of claim 1 , wherein the substrate surface between the zero mode waveguides comprises a polymer coating or resist.
7. The method of claim 1 , wherein density of the immobilized component on the bottom of the zero mode waveguides is 1000 times or more greater than density of the immobilized component on the substrate surface other than the bottom of the zero mode waveguides.
8. The method of claim 1 , wherein the polymerase component of the polymerase/template/primer complexes is bound to the bottom of the zero mode waveguides.
9. The method of claim 8 , wherein a single active polymerase molecule is bound to the bottom of each of a plurality of the zero mode waveguides.
10. The method of claim 8 , wherein the cladding layer comprises aluminum.