Nucleic acid purification with a binding matrix
View Patent ↗The present invention relates to methods, kits, and compositions for generating purified RNA samples and purified DNA samples. In particular, the present invention provides methods for generating a purified RNA or DNA sample from a sample containing both DNA and RNA molecules using a binding matrix that preferentially binds DNA or RNA in the presence of an acidic dilution buffer, or using a binding matrix that comprises acid zeolites, as well as compositions and kits for practicing such methods.
1. A method of generating a purified RNA sample from an initial sample that comprises DNA and RNA molecules, said method comprising:
a) contacting said initial sample with: i) a dilution buffer with an acidic pH, and ii) a nucleic acid binding matrix, which comprises a zeolite, that preferentially binds both single and double stranded DNA molecules in the presence of said dilution buffer, wherein said contacting generates a DNA-bound binding matrix which is substantially RNA free; and
b) physically separating said DNA-bound binding matrix from said initial sample, the remaining initial sample now being a purified RNA sample comprising a plurality of RNA molecules.
2. The method of claim 1 , further comprising step c) exposing said purified RNA sample to a binding component such that an RNA-bound binding component is generated which comprises a plurality of bound RNA molecules.
3. The method of claim 2 , further comprising step d) eluting at least a portion of said bound RNA molecules from said RNA-bound binding member with an elution solution such that a purified RNA preparation is generated, wherein said purified RNA preparation comprises a plurality of eluted RNA molecules.
4. The method of claim 1 , wherein said binding matrix is configured to not bind double stranded or single stranded RNA molecules.
5. The method of claim 4 , wherein said initial sample comprises a cell lysate, wherein said cell lysate comprises lysed cells, and wherein said plurality of eluted RNA molecules are present in said purified RNA preparation at a level of at least 5 μg of RNA per 1 million of said lysed cells present in said sample.
6. The method of claim 1 , wherein said purified RNA sample is substantially DNA-free.
7. The method of claim 4 , wherein said purified RNA preparation is substantially DNA-free.
8. The method of claim 4 , wherein said purified RNA sample or said purified RNA preparation contains less than 10 copies of a single copy gene per 100 ng of RNA in said initial sample.
9. The method of claim 1 , wherein said separating comprises passing said initial sample through a clearing column.
10. The method of claim 1 , wherein said separating comprises centrifuging said initial sample such that a pellet forms which contains said DNA-bound binding matrix, and separating said pellet from the remainder of said initial sample.
11. The method of claim 1 , wherein said binding matrix further comprises Fe 3 O 4 and said separating comprises magnetic separation of said DNA-bound binding matrix from said initial sample.
12. The method of claim 1 , wherein said sample is incubated at a temperature of between 25 and 80 degrees Celsius prior to step b).
13. The method of claim 1 , wherein said sample is incubated at a temperature of about 70 degrees Celsius prior to step b).
14. The method of claim 1 , wherein said dilution buffer comprises a citrate buffer, or both a citrate buffer and a chaotropic agent.
15. The method of claim 1 , wherein said sample comprises a cell lysate.
16. The method of claim 15 , wherein said cell lysate comprises nonnucleic acid cellular debris, and wherein separating said DNA-bound binding matrix serves to remove a substantial proportion of said non-nucleic acid cellular debris from said initial sample.
17. The method of claim 1 , wherein said binding matrix comprises binding particles, and said binding particles are in a composition comprising a salt solution.
18. The method of claim 1 , wherein said binding matrix comprises a membrane coated with binding particles.
19. The method of claim 18 , wherein said binding matrix comprises silicon.
20. The method of claim 1 , wherein said binding matrix comprises pores, wherein said pores are about 3 Å to 10 Å in size.
21. The method of claim 1 , wherein said dilution buffer has a pH of about 5.3 or less.
22. The method of claim 1 , wherein said DNA molecules in said sample comprise genomic DNA molecules.
23. The method of claim 1 , wherein the purified RNA sample contains less than 0.05% of the mass of DNA present in said initial sample.
24. A method of generating a purified RNA sample from an initial sample that comprises DNA and RNA molecules, said method comprising;
a) contacting said initial sample with; i) a dilution buffer with an acidic pH, and ii) a nucleic acid binding matrix comprising zeolite particles that preferentially binds both single and double stranded DNA molecules in the presence of said dilution buffer, wherein said contacting generates a DNA-bound binding matrix; and
b) physically separating said DNA-bound binding matrix from said initial sample the remaining initial sample now being a purified RNA sample comprising a plurality of RNA molecules which is substantially DNA free.
25. The method of claim 24 wherein the zeolite particles are selected from a group consisting of zeolite 3A and zeolite 13X.