Methods for the storage and synthesis of nucleic acids using a solid support
View Patent ↗The invention relates to storage of nucleic acid (particularly mRNA) on a solid support and to using such nucleic acid in nucleic acid synthesis or amplification reactions. In a preferred aspect, the invention provides synthesis of cDNA and cDNA libraries.
1. A method of producing one or more cDNA molecules comprising:
(a) contacting a sample comprising a cell or a virus with a solid medium, wherein:
(i) the sample comprises mRNA and genomic DNA;
(ii) the mRNA comprises an mRNA-template of interest; and
(iii) wherein the solid medium comprises:
a matrix; and
a composition for inhibiting degradation of the mRNA template, wherein:
the composition comprises a detergent or surfactant; and
the composition is sorbed to the matrix then dried prior to contact with the sample;
(b) sorbing at least a portion of the mRNA template to the solid medium;
(c) eluting the mRNA from the solid medium while retaining the genomic DNA; and
(d) contacting the mRNA with one or more reverse transcriptases under conditions sufficient to synthesize one or more cDNA molecules complementary to all or a portion of the mRNA template of interest.
2. The method of claim 1 , wherein the cDNA is a cDNA library.
3. The method of claim 1 , wherein the cDNA is double-stranded.
4. The method of claim 1 , further comprising:
(e) amplifying the cDNA.
5. The method of claim 4 , wherein the amplifying step comprises contacting at least one cDNA strand with a polymerase under conditions to synthesize one or more cDNA molecules complementary to all or a portion of the template.
6. The method of claim 4 , wherein the amplifying step comprises a polymerase chain reaction (PCR).
7. The method of claim 1 , wherein the detergent or surfactant of the composition is an anionic detergent or surfactant and wherein the composition further comprises:
(a) a base; and
(b) a chelating agent.
8. The method of claim 7 , wherein the composition further comprises uric acid or a urate salt.
9. The method of claim 7 , wherein:
(a) the anionic detergent or surfactant comprises sodium dodecyl sulfate (SDS);
(b) the base comprises Tris or tris-hydroxymethyl methane; and
(c) the chelating agent comprises ethylene diamine tetra-acetic acid (EDTA).
10. The method of claim 1 , wherein the matrix comprises a cellulose-based matrix or paper, or a micromesh of synthetic plastic material.
11. The method of claim 1 , wherein the matrix is selected from the group consisting of nitrocellulose, cellulose, diazocellulose, carboxymethylcellulose, hydrophilic polymers, polytetra-fluoro-ethylene, fiberglass, porous ceramics, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, agarose, agar, starch, and nylon.
12. The method of claim 1 , wherein the sample comprises a eukaryotic cell.
13. The method of claim 1 , wherein the eluting step further comprises contacting the solid medium comprising the mRNA with an elution buffer, wherein the elution buffer comprises:
(a) a base;
(b) a chelating agent;
(c) dithiothreitol; and
(d) a ribonuclease inhibitor.
14. The method of claim 13 , wherein the elution buffer further comprises:
(e) glycogen.
15. The method of claim 13 , wherein:
(a) the base comprises Tris or tris-hydroxymethyl methane; and
(b) the chelating agent comprises ethylene diamine tetra-acetic acid (EDTA).
16. The method of claim 13 , wherein the eluting step further comprises incubating the solid medium comprising the mRNA with the elution buffer at a temperature at or below 4° C.
17. The method of claim 13 , wherein the eluting step further comprises vortexing the solid medium comprising the mRNA with the elution buffer.