Nucleic acid sample preparation
The present invention includes methods, devices and systems for isolating a nucleic acid from a fluid comprising cells. In various aspects, the methods, devices and systems may allow for a rapid procedure that requires a minimal amount of material and/or results in high purity nucleic acid isolated from complex fluids such as blood or environmental samples.
1. A method of isolating a nucleic acid from a fluid comprising cells, comprising
a. applying the fluid to a device, the device comprising an array of electrodes capable of generating an AC electrokinetic field, wherein the first AC electrokinetic region is a first dielectrophoretic field region;
b. concentrating a plurality of cells in a first AC electrokinetic field region;
c. isolating nucleic acid in a second AC electrokinetic field region, wherein the second AC electrokinetic region is a second dielectrophoretic field region;
d. flushing the plurality of cells away in the first AC electrokinetic field;
e. performing PCR amplification on the nucleic acid, or a cDNA version of the nucleic acid, to produce a PCR product; and
f. isolating the PCR product in a third AC electrokinetic region.
2. The method of claim 1 , wherein the third AC electrokinetic region is a dielectrophoretic field region.
3. The method of claim 1 , wherein the third AC electrokinetic region is a dielectrophoretic high field region.
4. The method of claim 1 , wherein the array of electrodes is in a wavy or nonlinear line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a linker, wherein the dots and linker define the boundaries of the electrode, wherein the linker tapers inward towards or at the midpoint between the pair of dots, wherein the diameters of the dots are the widest points along the length of the repeating unit, wherein the edge to edge distance between a parallel set of repeating units is equidistant, or roughly equidistant.
5. The method of claim 1 , wherein the PCR amplification is performed in situ in the device.
6. The method of claim 1 , further comprising performing Sanger chain termination reactions on the PCR product to produce a sequencing product of the PCR-amplified nucleic acid.
7. The method of claim 6 , wherein the sequencing product of the PCR-amplified nucleic acid are separated electrophoretically.
8. The method of claim 7 , wherein the electrophoretic separation of the sequencing product of the PCR-amplified nucleic acid is capillary electrophoresis.
9. The method of claim 7 , further comprising the use of multicolor fluorescence detection to analyze the sequencing product of the PCR-amplified nucleic acid.
10. The method of claim 7 , wherein all steps are performed on a single chip.
11. The method of claim 1 , wherein the fluid comprising cells comprises no more than 10,000 cells.
12. A method of isolating a nucleic acid from a fluid comprising cells, the method comprising:
a. applying the fluid to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region;
b. concentrating a plurality of cells in a first AC electrokinetic field region, wherein the first AC eletrokinetic field region is a first dielectrophoretic high field region;
c. isolating the nucleic acid in a second AC electrokinetic field region, wherein the second AC electrokinetic field is a second dielectrophoretic high field region;
d. flushing the plurality of cells away in the first AC electrokinetic field;
e. performing PCR amplification on the nucleic acid, or a cDNA version of the nucleic acid, to produce a PCR product; and
f. isolating the PCR product in a third AC electrokinetic region.
13. The method of claim 12 , wherein the third AC electrokinetic region is a dielectrophoretic field region.
14. The method of claim 12 , wherein the third AC electrokinetic region is a dielectrophoretic high field region.
15. The method of claim 12 , wherein the array of electrodes is in a wavy or nonlinear line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a linker, wherein the dots and linker define the boundaries of the electrode, wherein the linker tapers inward towards or at the midpoint between the pair of dots, wherein the diameters of the dots are the widest points along the length of the repeating unit, wherein the edge to edge distance between a parallel set of repeating units is equidistant, or roughly equidistant.
16. The method of claim 12 , wherein the PCR amplification is performed in situ in the device.
17. The method of claim 12 , further comprising performing Sanger chain termination reactions on the PCR product to produce a sequencing product of the PCR-amplified nucleic acid.
18. The method of claim 17 , wherein the sequencing product of the PCR-amplified nucleic acid are separated electrophoretically.
19. The method of claim 18 , wherein the electrophoretic separation of the sequencing product of the PCR-amplified nucleic acid is capillary electrophoresis.
20. The method of claim 18 , further comprising the use of multicolor fluorescence detection to analyze the sequencing product of the PCR-amplified nucleic acid.
21. The method of claim 18 , wherein all steps are performed on a single chip.
22. The method of claim 12 , wherein the fluid comprising cells comprises no more than 10,000 cells.
23. A method of isolating a nucleic acid from a fluid comprising cells, comprising
a. applying the fluid to a device, the device comprising an array of electrodes capable of generating an AC electrokinetic field region, wherein the first AC electrokinetic field is a first dielectrophoretic field region;
b. concentrating a plurality of cells in a first AC electrokinetic field region;
c. isolating nucleic acid in a second AC electrokinetic field region; wherein the second AC electrokinetic field is a second dielectrophoretic field region;
d. flushing the plurality of cells away in the first AC electrokinetic field;
e. performing PCR amplification on the nucleic acid, or a cDNA version of the nucleic acid, to produce a PCR product;
f. isolating the PCR product in a third AC electrokinetic region;
g. performing Sanger chain termination reactions on the PCR product to produce a sequencing product of the PCR-amplified nucleic acid; and
h. performing electrophoretic separation of the sequencing product of the PCR-amplified nucleic acid.
24. The method of claim 23 , wherein the third AC electrokinetic region is a dielectrophoretic field region.
25. The method of claim 23 , wherein the third AC electrokinetic region is a dielectrophoretic high field region.
26. The method of claim 23 , wherein the array of electrodes is in a wavy or nonlinear line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of dots connected by a linker, wherein the dots and linker define the boundaries of the electrode, wherein the linker tapers inward towards or at the midpoint between the pair of dots, wherein the diameters of the dots are the widest points along the length of the repeating unit, wherein the edge to edge distance between a parallel set of repeating units is equidistant, or roughly equidistant.
27. The method of claim 23 , wherein the PCR amplification is performed in situ in the device.
28. The method of claim 23 , further comprising the use of multicolor fluorescence detection to analyze the sequencing product of the PCR-amplified nucleic acid.
29. The method of claim 23 , wherein all steps are performed on a single chip.
30. The method of claim 23 , wherein the fluid comprising cells comprises no more than 10,000 cells.