Methods for isolation of nucleic acids from prokaryotic spores
The invention provides methods and related products for extracting nucleic acids such as DNA from prokaryotic spores. The invention also encompasses methods for identifying the source of such spores via analysis of the isolated nucleic acids.
1. A method for harvesting high molecular weight nucleic acids from bacterial spores comprising
degrading spore coats of bacterial spores using a detergent and a reducing agent in a basic solution, thereby producing decoated spores,
lysing spore cell walls of the decoated spores using a solution comprising lysozyme and a chelator,
degrading proteins of the decoated spores using a solution comprising a protease and a mild detergent, and harvesting high molecular weight nucleic acids that are at least 30 kilobases in size.
2. The method of claim 1 , wherein the high molecular weight nucleic acids that are at least 30 kilobases in size are at least 250 kilobases in size.
3. The method of claim 1 , wherein the bacterial spores are Bacillus spores.
4. The method of claim 3 , wherein the Bacillus spores are B. anthracis spores.
5. The method of claim 1 , wherein the bacterial spores are Clostridium spores.
6. The method of claim 1 , wherein the bacterial spores are non-germinating.
7. A method for harvesting nucleic acids from bacterial spores comprising
contacting bacterial spores with a decoating solution for a time that is from about 5 minutes to less than 45 minutes at a temperature greater than 75° C. to degrade spore coats of the bacterial spores, thereby producing decoated spores,
sequentially contacting decoated spores with a first lysing solution comprising lysozyme and a second lysing solution comprising a protease for sufficient times under conditions sufficient to degrade spore cell walls and proteins of the decoated spores, and
harvesting nucleic acids that are at least 30 kilobases in size.
8. The method of claim 7 , wherein the nucleic acids that are at least 30 kilobases in size are at least 250 kilobases in size.
9. The method of claim 7 , wherein the bacterial spores are Bacillus spores.
10. The method of claim 9 , wherein the Bacillus spores are B. anthracis spores.
11. The method of claim 7 , wherein the bacterial spores are Clostridium spores.
12. The method of claim 7 , wherein the bacterial spores are non-germinating.