Methods and devices for making arrays
The present invention relates to methods and devices for amplifying nucleic acid, and, in particular, amplifying so as to generate products on a surface without the use of emulsions. In a preferred embodiment, a plurality of groups of amplified product are generated on the surface, each group positioned in different (typically predetermined) locations on said surface so as to create an array.
1. A method of amplifying nucleic acid template, comprising:
a) providing:
i) a first element comprising a first surface;
ii) a second element comprising a second surface;
iii) a plurality of indentations, wherein said indentations are formed on said first surface without extending through the entire thickness of said first surface;
iv) a first solution comprising a plurality of first PCR primers; and
v) a second solution comprising templates, polymerase, dNTPs and primers, wherein said primers are second PCR primers;
b) contacting said first surface with said first solution under conditions such that at least a portion of said first PCR primers attach to at least a portion of said first surface so as to create a modified surface comprising attached molecules;
c) contacting said modified surface with said second solution;
d) positioning said second surface on top of said modified surface, so as to create a plurality of first regions defined by said indentations and second regions defined by contact of said surfaces, said first regions comprising unmasked attached molecules, and said second regions comprising masked attached molecules, t hereby making a device;
e) thermocycling said device under conditions such that at least a portion of said template is amplified by said unmasked attached molecules.
2. The method of claim 1 , whereby said positioning of step d) causes at least a portion of said second solution on said modified surface of said first element to move off of said modified surface and be removed from said modified surface.
3. The method of claim 1 , wherein said contacting of step b) results in substantially the entire surface being contacted with said solution.
4. The method of claim 1 , wherein said second PCR primers consist of reverse PCR primers.
5. The method of claim 1 , further comprising the step f) separating said first element from said second element.
6. A method amplifying nucleic acid template without the use of emulsions, comprising:
a) providing:
i) a first element comprising a first surface;
ii) a second element comprising a second surface;
iii) a plurality of indentations, wherein said indentations are formed on said first surface;
iv) a first solution comprising a plurality of first PCR primers; and
v) a second solution comprising templates, polymerase, dNTPs and primers, wherein said primers are second PCR primers;
b) contacting said first surface with said first solution under conditions such that at least a portion of said PCR primers attach to at least a portion of said first surface so as to create a modified surface comprising attached molecules;
c) contacting said modified surface with said second solution;
d) positioning said second surface on top of said modified surface, so as to create a plurality of first regions defined by said indentations and second regions defined by contact of said surfaces, said first regions comprising unmasked attached molecules, and said second regions comprising masked attached molecules, thereby making a device;
e) thermocycling said device under conditions such that at least a portion of said template is amplified by said unmasked attached molecules without the use of emulsions.
7. The method of claim 6 , wherein said first element comprises glass.
8. The method of claim 7 , wherein said first element is a microscope slide.
9. A method amplifying nucleic acid template without the use of emulsions, comprising:
a) providing:
i) a first element comprising a first surface;
ii) a second element comprising a second surface;
iii) a plurality of indentations, wherein said indentations are formed on said first surface without extending through the entire thickness of said first surface, said indentations being between 50 nanometers and 50 microns in diameter, and spaced on said first surface at between 1.2 and 2 times the diameter;
iv) a first solution comprising a plurality of first PCR primers; and
v) a second solution comprising templates, polymerase, dNTPs and primers, wherein said primers are second PCR primers;
b) contacting said first surface with said first solution under conditions such that at least a portion of said PCR primers attach to at least a portion of said first surface so as to create a modified surface comprising attached molecules;
c) contacting said modified surface with said second solution;
d) positioning said second surface on top of said modified surface, so as to create a plurality of first regions defined by said indentations and second regions defined by contact of said surfaces, said first regions comprising unmasked attached molecules, and said second regions comprising masked attached molecules, thereby making a device; and
e) thermocycling said device under conditions such that at least a portion of said template is amplified by said unmasked attached molecules without the use of emulsions, wherein said template is not amplified by said masked molecules.
10. The method of claim 9 , wherein said first element comprises glass.
11. The method of claim 10 , wherein said first element is a microscope slide.