DNA in the presence of gellan

• US 8,404,446 B2
• Filed: 10/23/2009
• Issued: 03/26/2013
• Est. Priority Date: 11/20/2002
• Status: Expired due to Fees

• Current Assignee: Shin-Etsu Chemical Company Limited
• Original Assignee: Shin-Etsu Chemical Company Limited
• Inventors: Armentrout, Richard W.
• Primary Examiner(s): Ford, Allison
• Assistant Examiner(s): FERNANDEZ, SUSAN EMILY
• Application Number:
  US12/604,660
• Publication Number:
  US 20100041105A1
• Time in Patent Office: 1,250 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/15 Gradients

Abstract

A method is provided for nucleic acid amplification with enhanced sensitivity. The method for enhanced sensitivity involves carrying out the amplification reaction in the presence of gellan. For instance, the method allows for the production of detectable amounts of PCR amplified DNA from at least 10 fold fewer target molecules than a comparable PCR reaction in absence of gellan.

Claims

1. A method for enzymatically amplifying a target nucleic acid or a fragment thereof, comprising

(a) providing a nucleic acid amplification reaction mixture that comprises (i) a target nucleic acid, and (ii) gellan at a concentration above 0.005 wt % based on the weight of water;

and

(b) subjecting the nucleic acid amplification reaction mixture of (a) to conditions suitable for amplifying the target nucleic acid or a fragment thereof, whereby the target nucleic acid or the fragment thereof is amplified.

2. The method according to claim 1 wherein the amplification reaction mixture comprises gellan at a concentration above 0.01 wt % based on the weight of water.

3. The method according to claim 1 wherein the amplification reaction mixture comprises gellan at a concentration above 0.05 wt % based on the weight of water.

4. The method according to claim 1 wherein the amplification reaction mixture comprises gellan at a concentration above 0.1 wt % based on the weight of water.

5. The method according to claim 1 wherein the amplification reaction mixture comprises gellan at a concentration above 0.125 wt % based on the weight of water.

6. The method according to claim 1 wherein the amplification reaction mixture comprises gellan at a concentration above 0.15 wt % based on the weight of water.

7. In a method for enzymatically amplifying nucleic acid, the improvement comprising performing the enzymatic amplification in the presence of gellan or gellan fragments at a concentration of at least 0.005 wt % based on the weight of water.

8. A method for enzymatically amplifying a target nucleic acid or a fragment thereof, comprising

(a) providing a nucleic acid amplification reaction mixture having a water-based liquid phase and a gellan gel matrix phase, wherein the liquid phase comprises a target nucleic acid and is entrapped in the gel matrix phase;

and (b) subjecting the reaction mixture to conditions suitable for amplifying the target nucleic acid or a fragment thereof, whereby the target nucleic acid or the fragment thereof is amplified.

9. The method according to claim 8 wherein the gellan is intact.

10. The method according to claim 8 wherein the gellan is digested into small fragments.

11. The method according to claim 8 wherein the reaction mixture comprises at least 220 molecules of the target nucleic acid.

12. The method according to claim 8 wherein the reaction mixture comprises at least 2000 molecules of the target nucleic acid.

13. The method according to claim 8, wherein said gel matrix comprises less than 0.01 % wt nucleic acid other than the target nucleic acid based on the weight of the gellan.

14. The method according to claim 8 wherein the target nucleic acid or a fragment thereof is amplified using a method selected from the group consisting of PCR, LCR, TAS, NASBA, 3SR, RACE, and one-sided PCR.

15. The method according to claim 8 further comprising isolating the target nucleic acid from cells grown on gellan-containing medium.