Method for nucleic acid quantitation

• US 20090142767A1
• Filed: 11/26/2008
• Published: 06/04/2009
• Est. Priority Date: 11/29/2007
• Status: Active Grant

• Current Assignee: Hitachi, Ltd.
• Original Assignee: Hitachi, Ltd.
• Inventors: Taniguchi, Kiyomi, Kambara, Hideki
• Granted Patent: US 8,389,246 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2545/101 with an internal standard/control

Abstract

It is intended to provide a novel convenient approach for DNA quantitative analysis that overcomes the disadvantages of conventional formulations. A standard DNA sample is prepared by introducing a single-base substitution into target DNA, and a predetermined amount thereof is mixed with a target DNA sample. The target and standard DNAs are amplified using the same primers designed to amplify a region comprising the single-base substitution site. To a hybridization product of a probe capable of binding to a site immediately before the single-base substitution site, ddATP, ddGTP, ddCTP, and ddTTP are sequentially added one by one to perform a complementary strand synthesis reaction. Luciferase reaction-induced luminescence derived from the formed pyrophosphoric acid is detected. The target DNA is quantitated from the amount of the detected luminescence and the amount of the added standard DNA sample.

Claims

1. A method for nucleic acid quantitation, comprising the following steps:

1) mixing a target DNA sample with a predetermined amount of a standard DNA sample having a single-base substitution introduced in at least a portion of the target DNA;

2) amplifying the mixed sample using primers designed to amplify a region comprising the single-base substitution site;

3) dissociating the amplified sample into single strands, hybridizing thereto a probe capable of binding to a site immediately before the single-base substitution site, and sequentially adding ddATP, ddGTP, ddCTP, and ddTTP one by one to the hybridization product to perform a complementary strand synthesis reaction;

4) detecting luciferase reaction-induced luminescence derived from pyrophosphoric acid formed through the complementary strand synthesis reaction; and

5) quantitating the target DNA from the amount of the detected luminescence and the amount of the standard DNA sample.

2. The method according to claim 1, wherein the steps 1) to 5) are performed on plural standard DNA samples differing in the substituted base.

3. The method according to claim 1, wherein the steps 1) to 5) are performed on plural standard DNA samples differing in concentration.

4. The method according to claim 1, wherein at least one of the primers is biotinylated, and the method further comprises the step of purifying the amplification product using streptavidin-conjugated beads.

5. The method according to claim 1, wherein the target DNA sample is a cDNA sample.

6. The method according to claim 1, wherein an anchor sequence is added in advance to the target DNA, and the standard DNA sample is prepared using primers, at least one of which has the single-base substitution introduced in this anchor sequence.

7. The method according to claim 1, wherein both the target and standard DNAs are in a free form.

8. The method according to claim 1, wherein at least one of the target and standard DNAs is immobilized on a solid phase surface.

9. The method according to claim 1, wherein the target DNA sample is immobilized on magnetic beads.