Systems and methods for rapid nucleic acid extraction, purification and analysis from bone and tooth
Disclosed are processes and kits for rapid nucleic acid extraction from a nucleic acid-containing material, such as a bone, tooth or semen sample. For bone and tooth process involves providing the nucleic acid-containing material in a form suitable for nucleic acid extraction, adding a lysis buffer to the nucleic acid-containing material to obtain a mixture, mixing the mixture in a manner equivalent for about 30 seconds or longer and separating the mixture by centrifugation to obtain a liquid supernatant. The liquid supernatant contains the extracted nucleic acids which can be used for analysis including STR profiling by conventional or rapid DNA analysis. For semen the processes and kits involve applying an appropriate amount of sperm disruptive agent.
1. A process for extracting nucleic acids from a bone or tooth sample, comprising of:
a. providing the sample grinded into powder or hammered into fragments;
b. adding a demineralization buffer to the nucleic acid-containing material to obtain a mixture wherein the demineralization buffer consists essentially of a 0.3-0.7 M EDTA solution without any other chemicals;
c. mixing the mixture vigorously;
d. incubating the sample and the demineralization buffer for less than 60 minutes for a bone sample, and for less than 180 minutes for a tooth sample; and
e. separating the mixture to obtain a liquid supernatant wherein the liquid supernatant contains the extracted nucleic acids.
2. The process of claim 1 wherein the separation step does not involve concentrating the nucleic acid.
3. The process of claim 1 , wherein the volume of demineralization buffer is 300-350 μl for every 500 mg of bone or tooth, or 150-220 μl for every 100-200 mg of bone or tooth, or 100-150 μl for less than 100 mg of bone or tooth.
4. The process of claim 1 wherein the sample is a decomposed bone or tooth sample.
5. A process for extracting nucleic acids from a bone or tooth sample, comprising:
a. providing the bone or tooth sample grinded into powder or hammered into fragments;
b. adding demineralization buffer consisting essentially of less than 500 μl of a 0.5 M EDTA solution without any other chemicals to the nucleic acid-containing material to obtain a mixture;
c. mixing the mixture vigorously;
d. incubating the mixture for at least one minute, but less than 10 minutes with or without subjecting the mixture to heat; and
e. separating the mixture to obtain a liquid supernatant without concentration;
wherein the liquid supernatant contains the extracted nucleic acids.
6. The process of claim 5 , wherein the volume of demineralization buffer is 300-350 μl for every 500 mg of bone or tooth, or 150-220 μl for every 100-200 mg of bone or tooth, or 100-150 μl for less than 100 mg of bone or tooth.
7. The process of claim 5 wherein the sample has a weight of less than 10 mg.
8. The process of claim 5 wherein the sample is a decomposed bone or tooth sample.
9. The process of claim 1 wherein the step of separating the mixture to obtain a liquid supernatant is achieved by centrifugation.
10. The process of claim 9 wherein the centrifugation is at a speed of 20,000 rcf and above for 2 minutes or longer.
11. The process of claim 1 , further comprising a step of subjecting a portion of the liquid supernatant to a nucleic acid analysis.
12. The process of claim 11 wherein the nucleic acid analysis comprises transferring the portion of the liquid supernatant to a DNA processing system.
13. The process of claim 1 wherein the step of mixing the mixture vigorously is carried out by vortexing the mixture for about 30 seconds or longer.
14. A process for determining STR profiles of nucleic acids from a bone or tooth sample, comprising:
a. providing the bone or tooth grinded into powder or hammered into fragments;
b. adding a demineralization buffer to the bone or tooth to obtain a mixture wherein the demineralization buffer consists essentially of a 0.3-0.7 M EDTA solution without any other chemicals;
c. mixing the mixture vigorously;
d. incubating the sample and the demineralization buffer for less than 60 minutes for a bone sample, and for less than 180 minutes for a tooth sample; and
e. separating the mixture to obtain a liquid supernatant; wherein the liquid supernatant contains the extracted nucleic acids; and
f. subjecting a portion of the liquid supernatant to a nucleic acid analysis to determine the STR profile of the nucleic acids from the bone or tooth.
15. The process of claim 14 , wherein the volume of demineralization buffer is 300-350 μl for every 500 mg of bone or tooth, or 150-220 μl for every 100-200 mg of bone or tooth, or 100-150 μl for less than 100 mg of bone or tooth.
16. The process of claim 14 wherein the sample is a decomposed bone or tooth sample.
17. The process of claim 14 , wherein the step of separating the mixture to obtain a liquid supernatant is achieved by centrifugation.
18. The process of claim 17 , wherein the centrifugation is at a speed of 20,000 rcf and above for 2 minutes or longer.
19. The process of claim 14 wherein the step of mixing the mixture vigorously is carried out by vortexing the mixture for about 30 seconds or longer.
20. The process of claim 14 , wherein the nucleic acid analysis comprises transferring the portion of the liquid supernatant to a DNA processing system.
21. The process of claim 20 wherein at least seven STR loci are integrated.
22. The process of claim 14 wherein the demineralization buffer is 0.5M EDTA.