Methods, compositions and devices for amplification of nucleic acids
View Patent ↗Methods, kits and compositions of matter are provided which allow amplification of nucleic acid or interest or DNA of interest, comprising the steps of isolating template nucleic acid or DNA from a biological sample using an alkaline extraction solution and directly adding the extract to a reaction mixture, under conditions to amplify the nucleic acids, wherein the isolated template nucleic acid or DNA in the alkaline extraction solution is not diluted or not neutralized prior to the addition to the reaction mixture.
1. A composition comprising:
(a) a mixture of deoxynucleotides (dNTPs), divalent cations, a DNA-amplification enzyme, and amplification primers; and
(b) a buffer comprising (i) a salt of ammonium and an anion of a weak acid and (ii) a dicarboxylic acid, wherein said buffer has a buffering capacity at a sufficiently low ionic strength to sufficiently neutralize an alkaline solution comprising template nucleic acid or DNA in order to allow the amplification enzyme to function, wherein said salt is ammonium pentaborate, and wherein the dicarboxylic acid is malic acid, or the dicarboxylic acid is oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, tartaric acid, fumaric acid, maleic acid or a mixture thereof.
2. A kit comprising the composition of claim 1 .
3. A device comprising a kit according to claim 2 , said device further comprising
a) a chamber for sampling of plant material;
b) a liquid reservoir comprising an alkaline extraction solution; and
c) channels to direct the alkaline extraction solution, upon activation, over the plant material into the mixture.
4. A cartridge comprising the kit of claim 2 , said cartridge further comprising
a) a first component, and a second component, fitting together to be closed;
b) said first and second components comprising fitting and corresponding chamber parts which, when the first and second component are aligned, form a chamber A, fit to receive a biological sample;
c) said first component further comprising, attached to it, a liquid reservoir comprising an alkaline solution; said liquid reservoir being connected to said chamber A via a channel;
d) said first component further comprising at least one chamber B connected to said chamber A by at least one channel;
e) said first component further comprising at least one chamber C connected to said at least one chamber B by at least one channel; and
f) said second component comprising a physical access to allow interaction with the liquid reservoir;
wherein said composition is contained within at least one said chamber B.
5. A method for amplifying a nucleic acid of interest or a DNA of interest comprising applying the composition of claim 1 to nucleic acid of interest or DNA of interest.
6. The method of claim 5 , wherein the amplification of DNA is an isothermal amplification process.
7. The method of claim 5 , wherein the template nucleic acid or DNA is included in an alkaline solution.
8. The method of claim 7 , wherein the template nucleic acid or DNA in the alkaline solution further comprises carrier DNA.
9. A method for amplification of a nucleic acid of interest or DNA of interest, comprising
a. isolating template nucleic acid or DNA from a plant sample using an alkaline extraction solution; and
b. amplifying said nucleic acid or DNA using the composition according to claim 1 wherein said isolated template nucleic acid or DNA in said alkaline extraction solution is not diluted or not neutralized prior to said step b) application.
10. The method of claim 9 , wherein the alkaline extraction solution comprises KOH, NaOH or LiOH or mixtures thereof.
11. The method of claim 9 , wherein said plant sample is a plant part, organ or tissue, a plant leaf, seed, seed powder, single seeds or seed chips.
12. The device of claim 3 , wherein said device is capable of detecting amplification of nucleic acids.
13. The cartridge of claim 4 , wherein the first component is a base component and wherein the second component is a cover component.
14. A method for amplifying a nucleic acid of interest or a DNA of interest comprising using the kit of claim 2 .
15. A method for amplifying a nucleic acid of interest or a DNA of interest comprising using the device of claim 3 .
16. A method for amplifying a nucleic acid of interest or a DNA of interest comprising using the cartridge of claim 4 .
17. The method of claim 9 , wherein said salt is ammonium pentaborate.
18. The method of claim 9 , wherein said alkaline extraction solution comprises KOH in a concentration of 25 mM to 100 mM.
19. The method of claim 9 , wherein said biological sample is processed without further mechanical maceration.
20. The composition of claim 1 , wherein the DNA-amplification enzyme is DNA I polymerase, Klenow polymerase, TaqI polymerase, a DNA polymerase with strand displacing properties, phi 29 polymerase, Bst polymerase, Csa polymerase, 96-7 polymerase, Bsm polymerase, or GspSSD polymerase.
21. The composition of claim 1 , wherein the composition is suitable for use with an isothermal amplification process.
22. The composition of claim 1 , wherein the composition is suitable for use with Loop-mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), Polymerase chain Reaction (PCR), or Nicking Enzyme Amplification Reaction (NEAR).
23. The composition of claim 1 , wherein the composition is suitable for use with LAMP.
24. The method of claim 5 , wherein the DNA-amplification enzyme is DNA I polymerase, Klenow polymerase, TaqI polymerase, a DNA polymerase with strand displacing properties, phi 29 polymerase, Bst polymerase, Csa polymerase, 96-7 polymerase, Bsm polymerase, or GspSSD polymerase.
25. The method of claim 5 , wherein the method for amplifying is LAMP, RPA, PCR, or NEAR.
26. The method of claim 5 , wherein the method for amplifying is LAMP.
27. The method of claim 9 , wherein the method for amplification is an isothermal amplification process.
28. The method of claim 9 , wherein the DNA-amplification enzyme is DNA I polymerase, Klenow polymerase, TaqI polymerase, a DNA polymerase with strand displacing properties, phi 29 polymerase, Bst polymerase, Csa polymerase, 96-7 polymerase, Bsm polymerase, or GspSSD polymerase.
29. The method of claim 9 , wherein the method for amplification is LAMP, RPA, PCR, or NEAR.
30. The method of claim 9 , wherein the method for amplification is LAMP.
31. The composition of claim 1 , wherein the dicarboxylic acid is malic acid.
32. The method of claim 9 , wherein the dicarboxylic acid is malic acid.