Kit for assessing mitochondrial toxicity

• US 7,718,790 B2
• Filed: 03/15/2007
• Issued: 05/18/2010
• 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:
  US11/686,499
• Publication Number:
  US 20070196824A1
• Time in Patent Office: 1,160 Days
• Field of Search: None
• US Class Current: 536/24.33
• 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

Processes and methods for the simultaneous quantification of nucleic acids in diseased cells that are based on real-time PCR are provided. The real-time PCR protocol is an excellent tool for reliable quantification of in vitro drug screening and evaluation protocols to determine the efficacy of potential anti-viral agents. Quantification using these simultaneous PCR cycle threshold (Ct) detection techniques during one-step real-time RT-PCR (Applied Biosystems, CA) eliminates the variability resulting from quantification of end-point RT-PCR products. In addition, the mitochondrial toxicity assay is an added tool to assess potential side-effects for these chemotherapeutic agents.

Claims

1. A kit for assessing mitochondrial toxicity of a compound, comprising

a mixture of oligonucleotides comprising

at least one first primer set that provides a first detectable signal on the occurrence of amplification of mitochondrial nucleic acid;

and

at least one second primer set that provides a second detectable signal on the occurrence of amplification of host nuclear nucleic acid;

wherein the second primer set comprises the primers of SEQ ID No. 1 and SEQ ID No. 2.

2. The kit according to claim 1 further comprising a probe of SEQ ID No. 3 comprising a reporter molecule and a quencher molecule.

3. The kit according to claim 2, wherein the reporter molecule is FAM and the quencher molecule is TAMRA.

4. The kit according to claim 1, wherein the first primer set comprises the primers of SEQ ID No. 19 and SEQ ID No. 20.

5. The kit according to claim 4 further comprising a probe of SEQ ID No. 21 comprising a reporter molecule and a quencher molecule.

6. The kit according to claim 5, wherein the reporter molecule is TET and the quencher molecule is TAMRA.

7. A kit for assessing mitochondrial toxicity of a compound, comprising

a mixture of oligonucleotides comprising

at least one first primer set that provides a first detectable signal on the occurrence of amplification of mitochondrial nucleic acid;

and

at least one second primer set that provides a second detectable signal on the occurrence of amplification of host nuclear nucleic acid;

wherein the first primer set comprises the primers of SEQ ID No. 19 and SEQ ID No. 20.

8. The kit according to claim 7, wherein the second primer set comprises the primers of SEQ ID No. 1 and SEQ ID No. 2.

9. The kit according to claim 8 further comprising a probe of SEQ ID No. 3 comprising a reporter molecule and a quencher molecule.

10. The kit according to claim 9, wherein the reporter molecule is FAM and the quencher molecule is TAMRA.

11. The kit according to claim 7, wherein the first primer set comprises the primers of SEQ ID No. 19 and SEQ ID No. 20.

12. The kit according to claim 11 further comprising a probe of SEQ ID No. 21 comprising a reporter molecule and a quencher molecule.

13. The kit according to claim 12, wherein the reporter molecule is TET and the quencher molecule is TAMRA.