Simultaneous isolation and quantitation of DNA

• US 6,673,631 B1
• Filed: 08/20/1999
• Issued: 01/06/2004
• Est. Priority Date: 01/21/1997
• Status: Expired due to Term

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Bitner, Rex M., Tereba, Allan M., Ekenberg, Steven J., Smith, Craig E., Koller, Susan C., Kephart, Daniel D.
• Primary Examiner(s): LACOURCIERE, KAREN A
• Application Number:
  US09/377,986
• Time in Patent Office: 1,600 Days
• Field of Search: 435/6, 435/91.1, 435/287.2, 436/501, 436/526, 436/527, 423/335, 536/25.42
• US Class Current: 436/526
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: Y10S 977/838 Magnetic property of nanomaterial
• CPC Class Codes: Y10S 977/904 for medical, immunological, body treatment, or diagnosis
• CPC Class Codes: Y10S 977/916 Gene therapy
• CPC Class Codes: Y10S 977/92 Detection of biochemical
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis
• CPC Class Codes: Y10S 977/96 Of magnetic property

Abstract

The present invention provides methods for isolating a defined quantity of DNA target material from other substances in a medium. The method may be carried out using a known quantity of a silica-containing solid support, such as silica magnetic particles, having a definable capacity for reversibly binding DNA target material, and DNA target material in excess of the binding capacity of the particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the DNA target material in a mixture of the medium and particles, and separating the complex from the mixture using external magnetic force. The DNA target material may then be eluted from the complex. The quantity of DNA target material eluted may be determined based on a calibration model. The methods of the present invention permit isolation of DNA target material which is within a known quantity range. The methods of the invention eliminate the step of quantitating purified biological samples prior to further processing, such as amplification, Short Tandem Repeat (STR) analysis, and DNA sequencing. Samples of the DNA target materials may be obtained from liquid or solid media, such as liquid blood or paper.

Claims

1. A method for isolating a defined quantity of a DNA target material from other material in a medium by:

a. providing a medium including the DNA target material;

b. providing a discrete quantity of porous silica magnetic particles capable of reversibly binding a definable quantity of the DNA target material, the particles having an average particle diameter of about 5.0 to 8.5 microns, a nitrogen BET surface area of about 18 to 55 microns gm and an acid leach resistance of less than about 7 ppm Fe₂O₃, the amount of DNA target material provided in step (a) being in excess of the binding capacity of the particles;

c. forming a complex of the silica magnetic particles and the DNA target material by combining the silica magnetic particles and the medium;

d. removing the complex with the DNA target material from the medium by application of an external magnetic field; and

e. separating the DNA target material of step (c) from the complex by eluting the DNA target material, whereby a defined quantity of the DNA target material is obtained.

2. The method of claim 1, wherein the silica magnetic particles are siliceous-oxide coated magnetic particles.

3. The method of claim 1 wherein the medium includes a chaotropic salt.

4. The method of claim 3 wherein the chaotropic salt comprises guanidine thiocyanate.

5. The method of claim 1, wherein the DNA target material provided in step (a) is the product of a polymerase chain reaction.

6. The method of claim 1 wherein the DNA target material is genomic DNA.

7. The method of claim 1 wherein the DNA target material is plasmid DNA.

8. The method of claim 6 further comprising analyzing the eluted genomic DNA in a DNA typing process.

9. The method of claim 1 wherein the medium is a solid support containing the DNA target material and wherein the DNA target material is isolated from the solid support prior to step (c) by combining the solid support with a mixture comprising a chaotropic salt.

10. The method of claim 9 wherein the solid support is a paper.

11. The method of claim 9 wherein the mixture is heated to a temperature of from about 60°

to about 100°

C.

12. The method of claim 1 further comprising sequencing at least a portion of the eluted DNA target material.

13. The method of claim 1 further comprising a step of washing the complex after removal from the medium, before eluting the DNA target material from the complex.

14. The method of claims 13, wherein the complex is washed using a wash solution comprising an alcohol and a salt.

15. The method of claim 1, wherein the DNA target material eluted in step (e) is eluted with water.

16. A method of isolating a defined quantity of a DNA target material from other materials in a medium comprising the steps of:

a. providing a medium containing the DNA target material;

b. providing a discrete quantity of porous silica magnetic particles with the capacity to reversibly bind a definable quantity of the DNA target material per milligram of particle, the particles having an average particle diameter of about 5.0 to 8.5 microns, a nitrogen BET surface area of about 18 to 55 microns gm and an acid leach resistance of less than about 7 ppm Fe₂O₃, the amount of DNA target material provided in step (a) being in excess of the binding capacity of the particles;

c. forming a mixture comprising the medium, the silica magnetic particles, and a chaotropic salt, wherein the chaotropic salt concentration in the mixture is sufficient to cause the DNA target material to adhere to the particles;

d. incubating the mixture until at least some of the DNA target material is adhered to the silica magnetic particles;

e. removing the silica magnetic particles and the adhered DNA target material from the mixture using an external magnetic force; and

f. eluting the DNA target material of step (e) from the silica magnetic particles by exposing the particles to an elution solution, whereby a defined quantity of the DNA target material is obtained.

17. The method of claim 16 wherein the DNA target material is genomic DNA.

18. The method of claim 16 wherein the DNA target material is plasmid DNA.

19. The method of claim 16 further comprising sequencing at least a portion of the eluted DNA target material.

20. The method of claim 16, wherein the chaotropic salt comprises guanidine thiocyanate.

22. The method of claim 16, further comprising a step of washing the silica magnetic particles after removal from the medium, before eluting the DNA target material from the particles.

23. The method of claim 22, wherein the particles are washed using a wash solution comprising an alcohol and a salt.

24. The method of claim 16 wherein the elution solution is water.

21. The method of 16, wherein the concentration of chaotropic salt in the mixture formed in step (c) is between about 0.1 M and 7 M.

25. A method of determining a calibration model for quantitating a DNA target material in a sample type of interest, the method comprising:

a. providing a first medium, wherein the first medium includes a first quantity of the sample type of interest;

b. providing a second medium, wherein the second medium includes a second quantity of the sample type of interest, wherein the second quantity is greater than the first quantity of the sample type of interest;

c. mixing a first discrete quantity of silica magnetic particles with the first medium, wherein the silica magnetic particles are capable of reversibly binding a first defined quantity of the DNA target material, thereby forming a first complex of the silica magnetic particles and the DNA target material from the first medium, the discrete quantity of the sample type of interest in the first medium containing DNA target material in excess of the binding capacity of the particles mixed with the first medium;

d. mixing a second discrete quantity of silica magnetic particles with the second medium, wherein the silica magnetic particles are capable of reversibly binding a second defined quantity of the DNA target material, thereby forming a second complex of the silica magnetic particles and the DNA target material from the second medium, the discrete quantity of the sample type of interest in the second medium containing DNA target material in excess of the binding capacity of the particles mixed with the second medium;

e. removing the first complex from the first medium and the second complex from the second medium by application of an external magnetic field;

f. separately eluting the DNA target material from the first complex and second complex, producing a first eluent of isolated DNA target material from the first complex and a second eluent of isolated DNA target material from the second complex;

g. determining the amount of DNA target material in the first and in the second eluent; and

h. repeating steps a-g with at least one additional discrete quantity of silica magnetic particles until the amount of DNA target material isolated from two different media is determined to be about the same, thereby identifying conditions wherein the DNA binding capacity of the silica magnetic particles is exceeded.

26. The method of claim 25 wherein the first discrete quantity of particles provided in step, (c) the second discrete quantity of particles provided in step (d) and the at least one additional discrete quantity of particles provided in step (h) are the same quantity of particles.

27. A method of isolating DNA target material from a solid support, the method comprising:

contacting the solid support containing the DNA target material with a chaotropic salt solution at a temperature of about 60°

C. to about 100°

C. thereby isolating at least a portion of the DNA target material from the solid support;

isolating a defined quantity of DNA target material by adding a discrete quantity of silica magnetic particles to the isolated DNA target material to form a complex, the amount of DNA target material isolated from the solid support being in excess of the binding capacity of the particles;

removing the complex with the DNA target material from the solution by application of an external magnetic field; and

separating the DNA target material from the complex by eluting the DNA target material, whereby a defined quantity of the DNA target material is obtained.

28. The method of claim 27 wherein the solid support is paper.

29. The method of claim 27 wherein the chaotropic salt solution comprises a chaotropic salt and a pH buffer.