PCR compatible nucleic acid sieving matrix

• US 20040045827A1
• Filed: 09/10/2003
• Published: 03/11/2004
• Est. Priority Date: 03/20/2000
• Status: Active Grant

• Current Assignee: Caliper Life Sciences Incorporated (Revvity, Inc.)
• Original Assignee: Caliper Technologies Corp (Revvity, Inc.)
• Inventors: Mehta, Tammy Burd
• Granted Patent: US 7,521,186 B2
• Time in Patent Office: Days
• US Class Current: 204/450
• 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 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.

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

3. The method of claim 2, wherein the polymer solution comprises about 0.35% polymer or less.

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

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

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

7. The method of claim 1, 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⁺⁺.

8. The method of claim 1, comprising mixing the PCR reaction components with the sieving medium in a microfluidic channel.

9. The method of claim 8, 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.

10. The method of claim 9, wherein separating comprises electrophoretically separating.