PCR compatible nucleic acid sieving medium

• US 20020012971A1
• Filed: 02/23/2001
• Published: 01/31/2002
• Est. Priority Date: 03/20/2000
• Status: Abandoned Application

• Current Assignee: Caliper Holdings Incorporated
• Original Assignee: Caliper Holdings Incorporated
• Inventors: Mehta, Tammy Burd
• Application Number:
  US09/792,297
• Publication Number:
  US 20020012971A1
• Time in Patent Office: Days
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/101 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2565/137 Chromatographic separation
• CPC Class Codes: C12Q 2565/629 being a microfluidic device

Abstract

Sieving mediums comprising less than about 0.5% polymer, less than about 0.4% polymer, and 0.35% polymer or less are used to perform nucleic acid separations and PCR. The low polymer concentration does not inhibit PCR reactions and is sufficient for performing nucleic acids separations. Microfluidic devices are used to perform nucleic acids separations and PCR reactions in the sieving mediums described.

Claims

1. A microfluidic device for performing PCR and nucleic acid separations, the device comprising at least one microscale channel and a sieving medium, which sieving medium is disposed within the at least one microscale channel and comprises a polymer solution, which polymer solution comprises less than about 0.5% polymer.

2. The microfluidic device of claim 1, wherein the polymer solution comprises less than about 0.4% polymer.

3. The microfluidic device of claim 1, wherein the polymer solution comprises about 0.35% polymer or less.

4. The microfluidic device of claim 1, wherein the polymer solution comprises acrylamide.

5. The microfluidic device of claim 4, wherein the acrylamide comprises linear acrylamide, polyacrylamide, polydimethylacrylamide, or polydimethylacrylamide/coacrylic acid.

6. The microfluidic device of claim 1, wherein the polymer solution comprises agarose, methyl cellulose, polyethylene oxide, hydroxycellulose, or hydroxy ethyl cellulose.

7. The microfluidic device of claim 1, further comprising one or more proteins, nucleic acids, PCR reaction components, or PCR products disposed within the at least one microfluidic channel.

8. The microfluidic device of claim 7, wherein the PCR reaction components comprise one or more of:

a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, a primer which hybridizes to the nucleic acid template, or Mg⁺⁺.

9. A method of separating polynucleotides, the method comprising:

(i) providing two or more polynucleotides;

(ii) providing a sieving medium, which sieving medium comprises a polymer solution, which polymer solution comprises less than about 0.5% polymer;

(iii) allowing the two or more polynucleotides to migrate through the sieving medium, thereby separating the two or more polynucleotides.

10. The method of claim 9, wherein the polynucleotides comprise one or more PCR products, RNA, or DNA.

11. The method of claim 9, wherein the polymer solution comprises less than about 0.4% polymer.

12. The method of claim 9, wherein the polymer solution comprises about 0.35% polymer or less.

13. The method of claim 9, wherein the polymer solution comprises acrylamide.

14. The method of claim 13, wherein the acrylamide comprises linear acrylamide, polyacrylamide, polydimethylacrylamide, or polydimethylacrylamide/coacrylic acid.

15. The method of claim 9, wherein the polymer solution comprises agarose, methyl cellulose, polyethylene oxide, hydroxycellulose, or hydroxy ethyl cellulose.

16. The method of claim 9, further comprising introducing the sieving medium into a microfluidic channel and allowing the two or more polynucleotides to migrate through the sieving medium in the microfluidic channel.

17. The method of claim 9, wherein the separating comprises electrophoretically separating the two or more polynucleotides.

18. A method of performing PCR and separating one or more PCR products, the method comprising:

(i) mixing one or more PCR reaction components with a sieving medium to provide a PCR sieving medium, wherein the sieving medium comprises a polymer solution, which polymer solution comprises less than about 0.5% polymer; and

(ii) thermocycling the PCR sieving medium to produce one or more PCR products; and

,

(iii) separating the one or more PCR products by flowing the one or more PCR products through the sieving medium.

19. The method of claim 18, wherein the polymer solution comprises less than about 0.4% polymer.

20. The method of claim 19, wherein the polymer solution comprises about 0.35% polymer or less.

21. The method of claim 18, wherein the polymer solution comprises acrylamide.

22. The method of claim 21, wherein the acrylamide comprises linear acrylamide, polyacrylamide, polydimethylacrylamide, or polydimethylacrylamide/coacrylic acid.

24. The method of claim 18, wherein the one or more PCR reaction components comprise one or more of:

a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, a primer which hybridizes to the nucleic acid template, or Mg⁺⁺.

25. The method of claim 18, comprising mixing the PCR reaction components with the sieving medium in a microfluidic channel.

26. The method of claim 25, further comprising separating the one or more PCR products by flowing the one or more PCR products through the sieving medium in the microfluidic channel.

27. The method of claim 26, wherein separating comprises electrophoretically separating.

28. A nucleic acid sieving medium, which medium comprises one or more polynucleotides, one or more PCR reagents, and a polymer solution, which polymer solution comprises less than about 0.5% polymer.

23. The method of claim 28, wherein the polymer solution comprises agarose, methyl cellulose, polyethylene oxide, hydroxycellulose, or hydroxy ethyl cellulose.

29. The sieving medium of claim 28, wherein the polymer solution comprises less than about 0.4% polymer.

30. The sieving medium of claim 29, wherein the polymer solution comprises about 0.35% polymer or less.

31. The sieving medium of claim 28, wherein the polymer solution comprises acrylamide.

32. The sieving medium of claim 31, wherein the acrylamide comprises linear acrylamide, polyacrylamide, polydimethylacrylamide, or polydimethylacrylamide/coacrylic acid.

33. The sieving medium of claim 28, wherein the polymer solution comprises agarose, methyl cellulose, polyethylene oxide, hydroxycellulose, or hydroxy ethyl cellulose.

34. The sieving medium of claim 28, wherein the one or more PCR reaction components comprise one or more of:

a thermostable DNA polymerase, a plurality of nucleotides, a nucleic acid template, a primer which hybridizes to the nucleic acid template, or Mg⁺⁺.

35. The sieving medium of claim 28, wherein the one or more polynucleotides comprise DNA, RNA, or PCR products.