Methods of using dyes in association with nucleic acid staining or detection and associated technology

• US 8,232,050 B2
• Filed: 08/11/2010
• Issued: 07/31/2012
• Est. Priority Date: 03/17/2005
• Status: Active Grant

• Current Assignee: Biotium, Inc.
• Original Assignee: Biotium, Inc.
• Inventors: Mao, Fei, Leung, Wai-Yee
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/854,436
• Publication Number:
  US 20100323453A1
• Time in Patent Office: 720 Days
• Field of Search: 435/6, 536/23.1, 536/26.6
• US Class Current: 435/6
• CPC Class Codes: C09B 11/28 Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
• CPC Class Codes: C09B 15/00 Acridine dyes
• CPC Class Codes: C09B 23/04 one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Methods of using dyes and associated technology are provided. A dye, such as a monomeric dye or a dimeric dye, may be used in a nucleic acid gel staining application and/or a nucleic acid detection application. Such a dye and a salt that comprises an anion that is associated with a strong acid and a cation that is associated with a strong base may be used in such an application. A dimeric dye, such as a dimeric dye capable of forming a hairpin-like structure, may be used to stain and/or detect nucleic acids via a release-on-demand mechanism. A dimeric dye having low background fluorescence in the absence of nucleic acids and high fluorescence in the presence of nucleic acids, upon binding therewith, may be used to stain and/or detect nucleic acids.

Claims

1. A method of staining a sample comprising a nucleic acid immobilized on a surface or embedded in a gel matrix, the method comprising the steps of:

a) contacting the nucleic acid with an aqueous solution comprising a fluorescent nucleic acid dye and a salt at a concentration from about 5 mM to about 0.5 M , inclusive, wherein the salt comprises an anion that would be sufficient as a component of a strong acid and a cation that would be sufficient as a component of a strong base;

b) detecting a fluorescent signal associated with said fluorescent nucleic acid dye;

wherein the method produces a higher fluorescent signal than a corresponding method performed by contacting said nucleic acid with an aqueous solution lacking said salt.

2. The method of claim 1, wherein a concentration of the salt is about 0.05 M to about 0.2 M, inclusive.

3. The method of claim 1, wherein the strong acid has a pKa of about 2 or less and the strong base has a pKa of about 10 or more.

4. The method of claim 1, wherein the salt is selected from NaCl, sodium bromide, sodium sulfate, potassium chloride, potassium bromide, potassium sulfate, magnesium choride and tetramethylammonium chloride.

5. The method of claim 1, wherein the fluorescent nucleic acid dye is a monomeric dye or a dimeric dye.

6. The method of claim 1, wherein the dye is an asymmetric cyanine dye.

7. The method of claim 6, wherein the dye is SYBR®

Green.

8. The method of claim 5, wherein the dye is a dimeric dye.

9. The method of claim 8, wherein the dimeric dye has the formula: