Precircle probe nucleic acid amplification methods

• US 8,383,348 B2
• Filed: 08/09/2011
• Issued: 02/26/2013
• Est. Priority Date: 10/24/2000
• Status: Expired due to Term

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford Management Co.)
• Inventors: Willis, Thomas D., Hardenbol, Paul, Jain, Maneesh, Stolc, Viktor, Ronaghi, Mostafa, Davis, Ronald W.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US13/206,120
• Publication Number:
  US 20120065091A1
• Time in Patent Office: 567 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• 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

Abstract

The invention is directed to novel methods of multiplexing nucleic acid reactions, including amplification, detection and genotyping. The invention relies on the use of precircle probes that are circularized in the presence of the corresponding target nucleic acids, cleaved, and then amplified.

Claims

1. A method for detecting a target sequence comprising a first and second target domain in a sample, said method comprising:

a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising;

i) a first targeting domain;

ii) a second targeting domain;

iii) at least a first universal priming site; and

iv) a cleavage site;

wherein said first and second targeting domains hybridize to said first and second target domains;

b) contacting said first hybridization complex with a ligase to form a closed circular probe;

c) cleaving said closed circular probe at said cleavage site to form a cleaved probe;

d) amplifying said cleaved probe to form a plurality of amplicons; and

e) detecting said amplicons to detect the presence of said target sequence in said sample, wherein said sample is a purified sample.