Nucleic acid archiving
View Patent ↗This invention is directed to a process for tightly binding nucleic acid to solid phase and corresponding processes for the utilization thereof. Nucleic acid is bound to solid phase matrices exhibiting sufficient hydrophilicity and electropositivity to tightly bind the nucleic acids from a sample. These processes include nucleic acid (double or single stranded DNA and RNA) capture from high volume and/or low concentration specimens, buffer changes, washes, and volume reductions, and enable the interface of solid phase bound nucleic acid with enzyme, hybridization or amplification strategies. The tightly bound nucleic acid may be used, for example, in repeated analyses to confirm results or test additional genes in both research and commercial applications. Further, a method is described for virus extraction, purification, and solid phase amplification from large volume plasma specimens.
1. A method of amplifying one or more target nucleic acids, comprising:
a) contacting a sample containing said one or more target nucleic acids with a solid phase matrix and a buffer that allows said one or more target nucleic acid sequences to become bound to said matrix as single-stranded target nucleic acid, wherein said matrix is a specific binding material having one or more electropositive materials rendered hydrophilic;
b) contacting said matrix-bound target nucleic acid with a set of primer nucleic acid sequences and a buffer that allows said primer sequences to hybridize to said matrix-bound target nucleic acid; and
c) amplifying said one or more target nucleic acid to produce an amplified reaction mixture, wherein said target nucleic acid sequence remains bound to said matrix.
2. The method of claim 1 , wherein said buffer in step (b) reduces binding of said primer set to said solid phase matrix.
3. The method of claim 1 , wherein said electropositive material comprises elements selected from the group consisting of aluminum, titanium, zirconium, hafnium, scandium, yttrium, lanthanum, vanadium, tantalum, chromium, molybdenum, tungsten, boron, gallium, indium, germanium, tin, and lead.
4. The method of claim 3 , wherein said matrix is selected from the group consisting of aluminum oxide, titanium oxide (Ti 2 O 3 ), and modified zirconium dioxide (ZrO 2 ).
5. The method of claim 1 , wherein said matrix is selected from the group consisting of alpha aluminum oxide, gamma aluminum oxide and an aluminum oxide thin-film of mixed composition.
6. The method of claim 1 , wherein said matrix is Ti 2 O 3 .
7. The method of claim 1 , wherein said matrix is modified ZrO 2 .
8. The method of claim 1 , wherein said buffer in step (a) is selected from the group consisting of guanidine thiocyanate-based buffers, alkaline buffers, lithium chloride, and detergent based buffers.
9. The method of claim 1 , wherein said buffer in step (b) is a phosphate buffer.
10. The method of claim 1 , wherein said target nucleic acid is selected from the group consisting of double stranded DNA, single stranded DNA, RNA, or PNA.
11. The method of claim 1 , wherein said target nucleic acid is double stranded DNA and said buffer in step (a) allows said DNA to be bound to said matrix as single stranded DNA.
12. The method of claim 1 , wherein said amplification methodology is selected from the group consisting of PCR, SDA, NASBA, IsoCR, CRCA, Q beta replicase, branched chain DNA, RT-PCR, and unwinding coil amplification.
13. The method of claim 1 , further comprising repeating steps (b) and (c) one or more times.
14. The method of claim 1 , wherein said sample comprises two or more target nucleic acids and said two or more target nucleic acids are amplified in series.
15. The method of claim 1 , wherein said target nucleic acid contains multiple target nucleic acid sequences, said method further comprising contacting said matrix-bound target nucleic acid in step (b) with multiple primer sets to pre-amplify said multiple target sequences, wherein said multiple target sequences are amplified simultaneously.
16. The method of claim 15 , further comprising:
(d) dividing said pre-amplified reaction mixture of step (c) into a plurality of aliquots;
(e) adding at least one of said primer sets to each of said aliquots; and
(f) amplifying said aliquots.
17. The method of claim 1 , wherein said solid phase matrix is coated on the surface of a substrate.
18. The method of claim 17 , wherein said substrate is a glass or polymeric material.
19. The method of claim 17 , wherein said substrate is in the shape of tubes, plates, membranes, capillaries, slides, beads, microparticles, fibers, microchannels, and microarrays.