Simultaneous quantification of nucleic acids in diseased cells

• US 7,919,247 B2
• Filed: 12/03/2007
• Issued: 04/05/2011
• Est. Priority Date: 10/18/2000
• Status: Expired due to Fees

• Current Assignee: Gilead Pharmasset LLC (Gilead Sciences Inc.)
• Original Assignee: Pharmasset Incorporated (Gilead Sciences Inc.)
• Inventors: Stuyver, Lieven, Otto, Michael J.
• Primary Examiner(s): Johannsen; Diana B
• Application Number:
  US11/949,162
• Publication Number:
  US 20090220950A1
• Time in Patent Office: 1,219 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C07H 19/048 Pyridine radicals
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/142 Toxicological screening, e.g. expression profiles which identify toxicity
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

A process for assessing mitochondrial toxicity of a compound that includes contacting nucleic acids from a host with an amplification reaction mixture that contains at least two primers that provide detectable signals, wherein:

• • a first primer provides a first detectable signal upon amplification of a host mitochondrial nucleic acid;
  • a second primer provides a second detectable signal upon amplification of a host nuclear nucleic acid; and
  • comparing the first and second detectable signals.

Claims

1. A method for assessing mitochondrial toxicity of a compound in a living cell from a host, the method comprising:

contacting a living cell from a host with a compound;

recovering nucleic acids from the living host cell;

contacting the nucleic acids with an amplification reaction mixture, wherein the reaction mixture comprises

a first primer set, comprising the primers of SEQ ID No. 19 and SEQ ID No. 20, and a first probe that provides a first detectable signal upon amplification of a host mitochondrial nucleic acid;

a second primer set, comprising the primers of SEQ ID No. 1 and SEQ ID No. 2, and a second probe that provides a second detectable signal upon amplification of a host nuclear nucleic acid;

amplifying the host mitochondrial nucleic acid and host nuclear nucleic acid by RT-PCR; and

normalizing the first detectable signal to the second detectable signal and comparing the normalized first detectable signal from the living host cell that has been treated with the compound to a normalized first detectable signal from a control host cell that has not been treated with the compound, in order to assess the mitochondrial toxicity of the compound in the living host cell.

2. The method of claim 1 wherein the second probe comprises SEQ ID No. 3.

3. The method of claim 2 wherein the second probe comprises a reporter molecule and a quencher molecule.

4. The method of claim 3 wherein the reporter molecule is FAM and the quencher molecule is TAMRA.

5. The method of claim 1 wherein the first probe comprises SEQ ID No. 21.

6. The method of claim 5 wherein the first probe comprises a reporter molecule and a quencher molecule.

7. The method of claim 6 wherein the reporter molecule is FAM and the quencher molecule is TAMRA.

8. A method for assessing mitochondrial toxicity of an antiviral compound in a living cell from a host, the method comprising:

contacting a living cell from a host with an antiviral compound;

recovering nucleic acids from the living host cell;

contacting the nucleic acids with an amplification reaction mixture, wherein the reaction mixture comprises

a first primer set, comprising the primers of SEQ ID No. 19 and SEQ ID No. 20, and a first probe that provides a first detectable signal upon amplification of a host mitochondrial nucleic acid;

a second primer set, comprising the primers of SEQ ID No. 1 and SEQ ID No. 2, and a second probe that provides a second detectable signal upon amplification of a host nuclear nucleic acid;

amplifying the host mitochondrial nucleic acid and host nuclear nucleic acid by RT-PCR; and

normalizing the first detectable signal to the second detectable signal and comparing the normalized first detectable signal from the living host cell that has been treated with the antiviral compound to a normalized first detectable signal from a control host cell that has not been treated with the antiviral compound, in order to assess the mitochondrial toxicity of the antiviral compound in the living host cell.

9. The method of claim 8 wherein the second probe comprises SEQ ID No. 3.

10. The method of claim 9 wherein the second probe comprises a reporter molecule and a quencher molecule.

11. The method of claim 10 wherein the reporter molecule is FAM and the quencher molecule is TAMRA.

12. The method of claim 8 wherein the first probe comprises SEQ ID No. 21.

13. The method of claim 12 wherein the first probe comprises a reporter molecule and a quencher molecule.

14. The method of claim 13 wherein the reporter molecule is FAM and the quencher molecule is TAMRA.