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 for quantifying nucleic acid in a sample, the method comprising:
a. applying a sample with a conductivity of greater than 100 mS/m to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region, the device further comprising at least two chambers, the sample applied to a first chamber;
b. applying known quantities of a nucleic acid standard to a second chamber;
c. establishing a first AC electrokinetic high field region in the first chamber, the first AC electrokinetic high field capable of isolating larger nanoparticulate molecular targets;
d. establishing a second AC electrokinetic low field in the first chamber, the second AC electrokinetic low field capable of concentrating cells or micron-sized entities that may be present in the sample;
e. establishing a third AC electrokinetic high field to the second chamber, the second AC electrokinetic high field isolating the molecular nucleic acid standard applied to the second chamber;
f. flushing cells and micron-sized entities that may be present in the sample from the first chamber;
g. detecting bound nucleic acid signal on the array in the first chamber and second chamber; and
h. quantifying the bound nucleic acid by comparing the detected signal from the first chamber to detected signal from the second chamber.
2. The method of claim 1 , wherein the larger nanoparticulate molecular target is chosen from the group consisting of exosomes, high mw nucleic acids, including high mw DNA, oligo-nucleosome complexes, aggregated proteins, vesicle bound DNA, cell membrane fragments and cellular debris.
3. The method of claim 1 , further comprising detecting a target circulating cell-free biomarker, wherein the target circulating cell-free biomarker is chosen from the group consisting of mutations, deletions, rearrangements or methylated nucleic acid of circulating DNA, micro RNA, RNA from microvesicles or a combination thereof.
4. The method of claim 3 , wherein detection of the target circulating cell-free biomarker provides information useful for cancer diagnosis, cancer prognosis or treatment response in a patient.
5. The method of claim 4 , wherein the target circulating cell free biomarker is a tumor cell-free biomarker associated with CNS tumors, neuroblastoma, gliomas, breast cancer, endometrial tumors, cervical tumors, ovarian tumors, hepatocellular carcinoma, pancreatic carcinoma, esophageal tumors, Stoch tumors, colorectal tumors, head and neck tumors, nasopharyngeal carcinoma, thyroid tumors, lymphoma, leukemia, lung cancer, non-small cell lung carcinoma, small cell lung carcinoma, testicular tumors, kidney tumors, prostate carcinoma, skin cancer, malignant melanoma, squamous cell carcinoma or a combination thereof.
6. The method of claim 5 , wherein the hydrogel comprises two or more layers of a synthetic polymer.
7. The method of claim 5 , wherein the hydrogel has a viscosity between about 0.5 cP to about 5 cP prior to spin-coating.
8. The method of claim 5 , wherein the hydrogel has a conductivity between about 0.1 S/m to about 1.0 S/m.
9. The method of claim 5 , wherein the tumor cell-free biomarker is GFAP, VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9 or combinations thereof.
10. The method of claim 1 , further comprising detecting a target biomarker, wherein the target biomarker is chosen from the group consisting of proteins, lipids, antibodies, tumor cells and nanosomes.
11. The method of claim 1 , further comprising eluting the bound nucleic acid from the first chamber for further characterization.
12. The method of claim 11 , wherein the eluted nucleic acid is amplified or sequenced.
13. The method of claim 1 , wherein the sample is whole blood, serum, plasma, cerebrospinal fluid, body tissue, urine or saliva.
14. The method of claim 1 , wherein the sample is blood.
15. The method of claim 1 , wherein the AC electrokinetic field is produced using an alternating current having a voltage of 1 volt to 40 volts peak-peak, and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to 50%.
16. The method of claim 1 , wherein the conductivity of the fluid is greater than 500 mS/m.
17. The method of claim 1 , wherein the array of electrodes is spin-coated with a hydrogel having a thickness between about 0.1 microns and 1 micron.
18. The method of claim 1 , wherein the isolated nucleic acid comprises less than about 10% non-nucleic acid cellular material or cellular protein by mass.
19. The method of claim 1 , wherein the array of electrodes comprises a wavy line configuration, wherein the configuration comprises a repeating unit comprising the shape of a pair of dots connected by linker, wherein the linker tapers inward toward 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.
20. The method of claim 1 , wherein the array of electrodes comprises a passivation layer with a relative electrical permittivity from about 2.0 to about 4.0.
21. A method for analyzing a nucleic acid in a sample, the method comprising:
a. applying a sample with a conductivity of greater than 100 mS/m to a device, the device comprising an array of electrodes capable of establishing an AC electrokinetic field region, the device further comprising at least two chambers, the sample applied to a first chamber;
b. applying known quantities of a nucleic acid standard to a second chamber;
c. establishing a first AC electrokinetic high field region in the first chamber, the first AC electrokinetic high field capable of isolating larger nanoparticulate molecular targets;
d. establishing a second AC electrokinetic low field in the first chamber, the second AC electrokinetic low field capable of concentrating cells or micron-sized entities that may be present in the sample;
e. establishing a third AC electrokinetic high field to the second chamber, the second AC electrokinetic high field isolating the molecular nucleic acid standard applied to the second chamber;
f. flushing cells and micron-sized entities that may be present in the sample from the first chamber;
g. detecting bound nucleic acid signal on the array in the first chamber and second chamber;
h. eluting the bound nucleic acid from the first chamber; and
i. performing sequencing and/or polymerase chain reaction analysis on the eluted nucleic acid.
22. The method of claim 21 , wherein the larger nanoparticulate molecular target is chosen from the group consisting of exosomes, high mw nucleic acids, including high mw DNA, oligo-nucleosome complexes, aggregated proteins, vesicle bound DNA, cell membrane fragments and cellular debris.
23. The method of claim 21 , further comprising detecting a target circulating cell-free biomarker, wherein the target circulating cell-free biomarker is chosen from the group consisting of mutations, deletions, rearrangements or methylated nucleic acid of circulating DNA, micro RNA, RNA from microvesicles or a combination thereof.
24. The method of claim 23 , wherein detection of the target circulating cell-free biomarker provides information useful for cancer diagnosis, cancer prognosis or treatment response in a patient.
25. The method of claim 24 , wherein the target circulating cell-free biomarker is a tumor cell-free biomarker associated with CNS tumors, neuroblastoma, gliomas, breast cancer, endometrial tumors, cervical tumors, ovarian tumors, hepatocellular carcinoma, pancreatic carcinoma, esophageal tumors, Stoch tumors, colorectal tumors, head and neck tumors, nasopharyngeal carcinoma, thyroid tumors, lymphoma, leukemia, lung cancer, non-small cell lung carcinoma, small cell lung carcinoma, testicular tumors, kidney tumors, prostate carcinoma, skin cancer, malignant melanoma, squamous cell carcinoma or a combination thereof.
26. The method of claim 25 , wherein the tumor cell-free biomarker is GFAP, VEGF, EGFR, b-FGF, KRAS, YKL-40, MMP-9 or combinations thereof.
27. The method of claim 21 , further comprising detecting a target biomarker, wherein the target biomarker is chosen from the group consisting of proteins, lipids, antibodies, tumor cells and nanosomes.
28. The method of claim 21 , wherein the sample is whole blood, serum, plasma, cerebrospinal fluid, body tissue, urine or saliva.
29. The method of claim 21 , wherein the sample is blood.
30. The method of claim 21 , wherein the AC electrokinetic field is produced using an alternating current having a voltage of 1 volt to 40 volts peak-peak, and/or a frequency of 5 Hz to 5,000,000 Hz and duty cycles from 5% to 50%.