# US 2015/0104797 A1 · Composite visible colorant and method for quantitative amplification

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- Number: US 2015/0104797 A1
- Document ID: US20150104797A1
- Status: Published application
- Kind: A1
- Application number: 14/510,036
- Filed: 2014-10-08
- Published: 2015-04-16
- Earliest claimed: 2013-10-10
- Assignees: Bio-Rad Laboratories, Inc.
- Applicants: Bio-Rad Laboratories, Inc.
- Inventors: Rongdian Fu
- Web page: https://patentgrep.com/patent/US20150104797A1

## CPC

- C09K 11/06: containing organic luminescent materials
- C12Q 1/6804: Nucleic acid analysis using immunogens (immunoassay G01N33/53)
- C12Q 1/6844: Nucleic acid amplification reactions
- C12Q 1/6851: Quantitative amplification
- G01N 21/6408: {with measurement of decay time, time resolved fluorescence}
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C09K 2211/10: Non-macromolecular compounds
- C09K 2211/1018: Heterocyclic compounds
- C12Q 2563/107: fluorescence
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- C09K: MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Methods and compositions for using colorants with real-time amplification reactions and normalization passive dyes are provided.

## Claims

### Claim 1

A composite colorant formulation comprising:
a visually-detectable dye; and
an internal reference molecule comprising a first fluorophore, which first fluorophore upon excitation emits energy at a specific wavelength range;
wherein:
the visually-detectable dye partially absorbs energy at the specific wavelength range; and
the formulation does not comprise sufficient components for target nucleic acid amplification if added to a target nucleic acid and primers.

### Claim 2 (depends on 1)

The composite colorant formulation of claim 1, wherein the specific wavelength range is peaked at between 615 and 625 nm.

### Claim 3 (depends on 1, 2)

The composite colorant formulation of claim 1 or 2, wherein the first fluorophore is a 5- and/or 6-carboxy-X-rhodamine dye or its derivatives.

### Claim 4 (depends on 3)

The composite colorant formulation of claim 3, wherein the concentration of the 5- and/or 6-carboxy-X-rhodamine dye is more than 100 nM.

### Claim 5 (depends on 1)

The composite colorant formulation of claim 1, wherein the internal reference molecule comprises a second fluorophore and a linker joining the first and second fluorophores such that excitation of the second fluorophore results in transfer of energy from the second fluorophore to the first fluorophore, which first fluorophore in turn emits energy at the specific wavelength.

### Claim 6 (depends on 5)

The composite colorant formulation of claim 5, wherein the second fluorophore is fluorescein amidite (FAM).

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The composite colorant formulation of any of claims 1-6, wherein the visually-detectable dye is selected from the group consisting of Xylene Cyanol FF, Cresol Red, Tertrazine, Quinoline Yellow, m-Cresol Purple, Brilliant Blue, Patent Blue, Indigocarmine, Acid Red 1, Neutral Red, Bromocresol Green, Acid Violet 5, Bromo phenol blue, and Orange G.

### Claim 8 (depends on 1, 2, 3, 4, 5, 6, 7)

The composite colorant formulation of any of claims 1-7, wherein
the visually-detectable dye is at an integer multiple of a visually-detectable concentration; and
the internal reference molecule is at said integer multiple of a sufficient concentration to compensate for absorbance of a passive reference signal in a qPCR reaction by the visually-detectable dye at the specific wavelength range;
wherein said integer multiple is between 2 and 1000.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The composite colorant formulation of any of claims 1-8, further comprising a long Stokes-shift passive reference dye different from the internal reference molecule, wherein the long Stokes-shift dye has a Stokes-shift greater than the Stokes-shift of the first fluorophore, wherein the long Stokes-shift dye has an emission wavelength maximum approximately the same as the first fluorophore emission wavelength maximum, and an excitation wavelength maximum significantly different than the first fluorophore excitation wavelength maximum.

### Claim 10 (depends on 9)

The composite colorant formulation of claim 9, wherein the long Stokes-shift dye has a Stokes-shift of at least about 60 nm.

### Claim 11 (depends on 9)

The composite colorant formulation of claim 9, wherein the long Stokes-shift dye has a excitation wavelength maximum of 550 nm or less.

### Claim 12 (depends on 9)

The composite colorant formulation of claim 9, wherein said long Stokes-shift dye is a fluorescent dot.

### Claim 13 (depends on 1)

The composite colorant formulation of claim 1, wherein the internal reference molecule is a long Stokes-shift dye, wherein the long Stokes-shift dye has an emission wavelength maximum approximately the same as a 5- and/or 6-carboxy-X-rhodamine dye emission wavelength maximum, and an excitation wavelength maximum of about 550 nm or less.

### Claim 14

The composite colorant formulation of any of the above claims, wherein the formulation lacks a polymerase, nucleotides, or divalent cations.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

A method of forming a mixture capable of supporting nucleic acid amplification, the method comprising,
combining the composite colorant formulation of any of claims 1-14 with at least a DNA polymerase, nucleotides, divalent cations, a passive reference dye for amplification normalization and a nucleic acid sample.

### Claim 16 (depends on 15)

The method of claim 15, comprising combining the composite colorant formulation with a real-time PCR master mixture comprising at least a DNA polymerase, nucleotides, divalent cations, a passive reference dye for amplification normalization, thereby forming a colored real-time PCR master mixture; and
combining a nucleic acid sample with the colored real-time PCR master mixture.

### Claim 17 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

A method of performing a real-time quantitative polymerase chain reaction, the method comprising,
performing a polymerase chain reaction (PCR) with a mixture comprising the composite colorant formulation of any of claims 1-14 and at least a DNA polymerase, nucleotides, divalent cations, a passive reference dye for amplification normalization, and a nucleic acid sample.