Process and apparatus for fragmenting biomaterials

• US 5,610,010 A
• Filed: 03/07/1994
• Issued: 03/11/1997
• Est. Priority Date: 10/11/1990
• Status: Expired due to Term

• Current Assignee: Indiana University Research & Technology Corporation (Indiana University)
• Original Assignee: Indiana University Foundation (Indiana University)
• Inventors: Togasaki, Robert K., Surzycki, Stefan J., Kityama, Masahito
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): LEITH, NANCY JILL
• Application Number:
  US08/039,430
• Time in Patent Office: 1,100 Days
• Field of Search: 435/6, 435/283.1, 435/287, 435/285.1, 422/99, 422/100, 422/101, 422/102, 422/243, 422/244, 536/22.1, 239/338, 935/19, 530/350
• US Class Current: 435/6.12
• CPC Class Codes: B05B 7/0012 Apparatus for achieving spraying before discharge from the apparatus
• CPC Class Codes: C12M 47/06 Hydrolysis; Cell lysis; Extraction of intracellular or cell wall material lysis of microorganisms C12N1/06; extracting or separating nucleic acids from biological samples C12N15/1003
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: G01N 1/286 involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising microtomes G01N1/06; pulverising in general B02C; mixing in general B01F
• CPC Class Codes: G01N 2001/4066 using difference of solubility between liquid and gas, e.g. bubbling, scrubbing or sparging

Abstract

Described is an improved process for fragmenting a biomaterial and isolating and recovering a component thereof. The preferred improved process includes the step of performing the fragmentation by nebulizing a liquid medium containing the biomaterial. A preferred process for fragmenting isolated DNA includes the step of nebulizing a fluid containing the DNA. This preferred process provides randomness superior to prior known DNA fragmentation methods, as well as other important advantages. An improved nebulization device is also described.

Claims

1. In a process for fragmenting a biomaterial and isolating and recovering a component thereof, the improvement comprising performing said fragmenting by nebulizing a liquid containing said biomaterial, said nebulizing including directing the liquid containing the biomaterial against a nebulization barrier so as to cause the biomaterial to be fragmented.

2. A process according to claim 1, wherein said, nebulizing includes repeatedly nebulizing said liquid.

3. A process according to claim 1, and also including recovering said component in a substantially pure form.

4. A process according to claim 1, wherein said biomaterial is DNA, a starch polymer, or a cell.

5. A process according to claim 1, wherein said biomaterial is a cell and said component is a cell subfraction.

6. A process according to claim 5, wherein said cell subfraction is DNA, a protein or a cell organelle.

7. A process according to claim 6, wherein said cell subfraction is a protein.

8. A process according to claim 6, wherein said cell subfraction is DNA.

9. A process according to claim 6, wherein said cell subfraction is a cell organelle.

10. A process according to claim 9, wherein said cell organelle is a nucleus, mitochondrion, or a chloroplast.

11. A process for fragmenting isolated DNA, comprising the step of nebulizing a liquid containing said DNA wherein said nebulizing takes place in a device which has a liquid input and a liquid output, wherein said device also comprises means for returning the liquid from the output to the input.

12. A process according to claim 11, and also comprising the step of recovering said DNA after said nebulizing.

13. A process according to claim 12, and also including the step of repairing the ends of said DNA after said recovering.

14. A process according to claim 11, wherein said DNA is substantially pure.

15. A process according to claim 14, wherein said nebulizing includes repeatedly nebulizing said liquid.

16. A process according to claim 11, and also comprising the step of including an agent in said liquid to increase its viscosity.

17. A process according to claim 11, wherein said liquid includes water.

18. A process according to claim 17, wherein said viscosity-increasing agent is a sugar or an alcohol.

19. A process according to claim 18, wherein said viscosity-increasing agent is glycerol, ethylene glycol or sucrose.

20. A process according to claim 19, wherein said DNA is included in sufficient amounts whereby self-protection provides larger final DNA fragments.

21. A process according to claim 19, wherein a suitable polymer is included in said liquid in an amount whereby said DNA is protected and larger fragments are obtained.

22. A process for determining a sequence of a DNA strand, comprising the steps of (i) nebulizing a liquid containing multiple copies of the DNA strand to thereby form DNA fragments, said nebulizing including directing the liquid containing the copies of the DNA strand against a nebulization barrier so as to cause the copies of the DNA strand to be fragmented, and (ii) determining by analysis of said fragments, a sequence of said DNA strand.

23. A process for producing DNA fragments, comprising repeatedly nebulizing liquid containing DNA, said nebulizing including directing the liquid containing DNA against a nebulization barrier, to thereby produce DNA fragments of ±

200 base pairs at an efficiency of at least about 30%.

24. A process according to claim 23, wherein said efficiency is at least about 50%.

25. In a process for fragmenting a cell, the improvement comprising performing said fragmenting by nebulizing a liquid containing said cell, said nebulizing including directing the liquid containing the cell against a nebulization barrier so as to cause the cell to be fragmented.

26. A process according to claim 25 further comprising recovering a component of the cell in a substantially pure form.

27. A process according to claim 26, wherein said component is a protein.

28. A process according to claim 27, wherein said component is DNA.

29. A process according to claim 28, wherein said component is a cell organelle.