Homogeneous assay system

• US 6,214,979 B1
• Filed: 09/19/1997
• Issued: 04/10/2001
• Est. Priority Date: 08/06/1990
• Status: Expired due to Fees

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Roche Molecular Systems (Roche Holding AG)
• Inventors: Holland, Pamela M., Watson, Robert M., Gelfand, David H., Saiki, Randall K.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/934,378
• Time in Patent Office: 1,299 Days
• Field of Search: 435/6, 435/810, 436/501, 935/77, 935/78, 536/22.1, 536/23.1, 536/24.3
• US Class Current: 536/22.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds

Abstract

A process of detecting a target nucleic acid using labeled oligonucleotides uses the 5′ to 3′ nuclease activity of a nucleic acid polymerase to cleave annealed labeled oligonucleotide from hybridized duplexes and release labeled oligonucleotide fragments for detection. This process is easily incorporated into a PCR amplification assay.

Claims

1. A detectably labeled oligonucleotide probe, which probe is blocked at the 3′

terminus to prohibit polymerase catalyzed extension of said probe, wherein said blocking is achieved either by adding a chemical moiety to the 3′

hydroxyl of the terminal nucleotide, which chemical moiety does not also serve as a label for subsequent detection, or by removing said 3′

hydroxyl; and

wherein said labeled oligonucleotide probe comprises a pair of non-radioactive interactive labels consisting of a first label and a second label, said first label and second label attached to said oligonucleotide directly or indirectly, and wherein said first label is separated from said second label by a nuclease susceptible cleavage site.

2. The detectably labeled oligonucleotide probe of claim 1 where in said first label is at the 5′

terminus and said second label is at the 3′

terminus.

3. The detectably labeled oligonucleotide probe of claim 1 wherein said first and second labels comprise a pair of interactive signal-generating labels positioned on said labeled oligonucleotide to quench the generation of detectable signal.

4. The detectably labeled oligonucleotide probe of claim 3 wherein said first label is a fluorophore and said second label is a quencher which interacts therewith.