Solid matrix for one step nucleic acid amplification
View Patent ↗The present invention to relates to methods and kits which can be used to amplify nucleic acids with the advantage of decreasing user time and possible contamination. The PCR reagents are bound to a solid matrix for easy processing and amplification of DNA samples.
1. A solid matrix for storing and/or amplification of nucleic acids comprising a solid matrix, wherein the matrix comprises a lysis reagent and a sequestering reagent, and wherein the matrix comprises both the lysis reagent and the sequestering reagent before the matrix has any contact with a nucleic acid.
2. The solid matrix of claim 1 , wherein said lysis reagent comprises an anionic surfactant or detergent.
3. The solid matrix of claim 2 , wherein said anionic surfactant is sodium dodecyl sulphate (SDS).
4. The solid matrix of claim 1 , wherein said sequestering agent is a cyclodextrin.
5. The solid matrix of claim 4 , wherein the cyclodextrin is selected from the group consisting of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.
6. The solid matrix of claim 4 , wherein the cyclodextrin is α-cyclodextrin.
7. The solid matrix of claim 4 , wherein the cyclodextrin is selected from the group consisting of 6-O-α-D-Maltosyl-β-cyclodextrin, hydroxyethyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin.
8. The solid matrix of claim 1 , wherein the solid matrix comprises a glass or silica-based solid phase medium, a plastics-based solid phase medium or a cellulose-based solid phase medium.
9. The solid matrix of claim 8 , wherein the solid phase medium is selected from one of the following: glass, glass fiber, glass microfiber, silica, silica gel, silica oxide, cellulose, nitrocellulose, carboxymethylcellulose, polyester, polyamide, carbohydrate polymers, polypropylene, polytetrafluorethylene, polyvinylidinefluoride, wool or porous ceramics.
10. The solid matrix of claim 1 , wherein the solid matrix is a cellulose based matrix.
11. The solid matrix of claim 10 , wherein said cellulose based matrix is in the form of a pre punched disc.
12. The solid matrix of claim 10 , wherein said cellulose based matrix is in the form of a pre punched disc having a cellulose based matrix and comprising a protein denaturing agent and a chelating agent.
13. The solid matrix of claim 1 , wherein the sequestering reagent and the lysis reagent are separated physically in the matrix.
14. The solid matrix of claim 13 , wherein the lysis reagent is adjacent to the sequestering reagent on said solid matrix.
15. The solid matrix of claim 13 , wherein the sequestering reagent and the lysis reagent are separated by a spacer.
16. The solid matrix of claim 1 , further comprising lyophilised or impregnated or printed Ready to Go PCR reagents for one step nucleic acid amplification, wherein the Ready to Go PCR reagents comprise a DNA polymerase, a deoxyribonucleotide triphosphate (dNTP), a reaction buffer, and at least one primer.
17. The solid matrix of claim 16 , wherein the lysis reagent, the sequestering reagent, and the Ready to Go PCR reagents are physically separated by a spacer.
18. A method for amplification of nucleic acid using polymerase chain reaction comprising the steps:
i) contacting the solid matrix of claim 1 with a cellular sample containing nucleic acid;
ii) transferring the solid matrix to a reaction vessel; and
iii) incubating the nucleic acid with a PCR reagent mixture for amplification of the nucleic acid.
19. The method of claim 18 , wherein said PCR reagent mixture comprises a DNA polymerase, a deoxyribonucleotide triphosphate (dNTP), a reaction buffer, and at least one primer.
20. A method for amplification of nucleic acid using polymerase chain reaction comprising the steps:
i) contacting the solid matrix of claim 16 with a cellular sample containing nucleic acid;
ii) transferring the solid matrix to a reaction vessel; and
iii) incubating nucleic acid with liquid water and forward and reverse primers for amplification of the nucleic acid.
21. A method of detecting amplified nucleic acid using a detection system comprising the steps:
i) amplifying nucleic acids using the method of claim 18 ; and
ii) detecting the amplified nucleic acids with a computer.
22. The method of claim 21 , wherein the detecting step is performed using a PCR imaging system.
23. The method of claim 18 , wherein the cellular sample is selected from a group comprising a virus, a bacteria and tissue culture cells.
24. A kit for storing and amplifying nucleic acid comprising the solid matrix of claim 1 and instructions for use thereof.
25. A kit for storing and amplifying nucleic acid comprising the solid matrix of claim 16 and instructions for use thereof.
26. A method of detecting the amplified nucleic acid using a detection system comprising the steps:
i) amplifying nucleic acids using the method of claim 20 ; and
ii) detecting the amplified nucleic acid with a computer.
27. The method of claim 20 , wherein the cellular sample is selected from a group comprising a virus, a bacteria and tissue culture cells.