Aqueous compositions that maintains fidelity of the nucleic acid sequences of a biological specimen
Disclosed are compositions for isolating populations of nucleic acids from biological, forensic, and environmental samples. Also disclosed are methods for using these compositions as one-step formulations for killing pathogens, inactivating nucleases, and releasing polynucleotides from other cellular components within the sample, and stabilizing the nucleic acids prior to further processing or assay. The disclosed compositions safely facilitate rapid sample collection, and provide extended storage and transport of the samples at ambient or elevated temperature without contamination of the sample or degradation of the nucleic acids contained therein. This process particularly facilitates the collection of specimens from remote locations, and under conditions previously considered hostile for preserving the integrity of nucleic acids released from lysed biological samples without the need of refrigeration or freezing prior to molecular analysis.
1. An aqueous composition that maintains fidelity of a nucleic acid sequence contained with a biological sample when mixed with the sample and maintained for a period of time without refrigeration, comprising components that upon mixing sterilize the sample, and denature proteins and inactivate nucleases of the sample, wherein the composition comprises guanidine thiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris), ethylenediaminetetraacetic (EDTA), and nuclease-free water in amounts such that the fidelity of the nucleic acid sequence as determined by PCR is within three CTs within the period of time, and the period of time is at least 7 days.
2. The composition of claim 1 , wherein the sample is selected from the group consisting of blood, plasma, serum, sputum, urine, stool, buccal swabs, throat swabs, vaginal swabs, urethral swabs, cervical swabs, rectal swabs, lesion swabs, abscess swabs, nasopharyngeal swabs, tears, mucus, saliva, semen, vaginal fluids, lymphatic fluid, amniotic fluid, spinal fluid, cerebrospinal fluid, peritoneal effusions, pleural effusions, exudates, punctates, epithelial smears, biopsies, bone marrow samples, fluid from a cyst or abscess, synovial fluid, vitreous or aqueous humor, eye washes or aspirates, pulmonary lavage or lung aspirates, an organ or tissue, and any combination thereof.
3. The composition of claim 1 , wherein the composition comprises the guanidine thiocyanate in an amount from about 0.5 M to about 6M, the TCEP is in an amount from about 0.5 mM to about 30 mM, the N-lauroyl sarcosine in an amount from about 0.15% to about 1% (wt./vol.), the sodium citrate in an amount from about 0.5 mM to about 50 mM, and the EDTA in an amount from about 0.1 mM to about 1.0 mM, and the Tris comprises from about 1 mM to about 1 M.
4. The composition of claim 3 , wherein the composition further comprises ethanol.
5. The composition of claim 4 , wherein the composition comprises the silicone polymer in an amount from about 0.0001% to about 0.3% (wt./vol.), and the ethanol in an amount from about 1% to about 25% (vol./vol.).
6. The composition of claim 1 , wherein the period of time is at least 14 days.
7. The composition of claim 1 , wherein the period of time is at least 21 days.
8. The composition of claim 1 , wherein the period of time is at least 28 days.
9. The composition of claim 1 , wherein the period of time is several months.
10. The composition of claim 1 , which is transported for analyzing fidelity of nucleic acids.
11. The composition of claim 1 , wherein the fidelity of the nucleic acid sequence is within two CTs.
12. The composition of claim 1 , wherein the fidelity of the sequence is within three CTs for an RNA sequences and/or within two CTs for a DNA sequence.
13. The mixture comprising the composition of claim 1 and a biological sample.
14. The mixture of claim 13 , which is safe for handling and transportation.
15. The mixture of claim 13 , which is maintained at room temperature.
16. The mixture of claim 13 , wherein room temperature is from 15° C. to 30° C.
17. The mixture of claim 13 , wherein room temperature is from 18° C. to 28° C.
18. An aqueous composition that maintains fidelity of a nucleic acid sequence contained with a biological sample when mixed with the sample and maintained for a period of time without refrigeration, comprising components that upon mixing sterilize the sample, and denature proteins and inactivates nucleases of the sample, wherein the composition comprises guanidine thiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris), ethylenediaminetetraacetic (EDTA), and nuclease-free water in amounts such that the fidelity of the nucleic acid sequence as determined by PCR is within three CTs within the period of time for an RNA sequence and within two CTs for a DNA sequence within the period of timeand the period of time is at least 7 days.
19. The composition of claim 18 , wherein the period of time is at least 14 days.
20. The composition of claim 18 , wherein the period of time is at least 21 days.
21. The composition of claim 18 , wherein the period of time is at least 28 days.
22. The composition of claim 18 , wherein the period of time is several months.
23. The composition of claim 18 , wherein the pH of the composition is from about 6 to about 7.
24. The composition of claim 18 , wherein the composition comprises the guanidine thiocyanate in an amount from about 0.5 M to about 6M, the TCEP is in an amount from about 0.5 mM to about 30 mM, the N-lauroyl sarcosine in an amount from about 0.1% to about 1% (wt./vol.), the sodium citrate in an amount from about 0.5 mM to about 0.3 M, and the EDTA in an amount from about 0.5 mM to about 50 mM.
25. An aqueous composition comprising guanidine thiocyanate, tris(2-carboxyethyl) phosphine (TCEP), sodium citrate, N-lauroyl sarcosine, a silicone polymer, tris(hydroxymethyl) aminomethane (Tris), ethylenediaminetetraacetic (EDTA), and nuclease-free water, wherein the pH of the composition is from about 6 to about 7.
26. The composition of claim 25 , wherein the composition comprises the guanidine thiocyanate in an amount from about 0.5 M to about 6M, the TCEP is in an amount from about 0.5 mM to about 30 mM, the N-lauroyl sarcosine in an amount from about 0.1% to about 1% (wt./vol.), the sodium citrate in an amount from about 0.5 mM to about 0.3 M, and the EDTA in an amount from about 0.5 mM to about 50 mM.