Precircle probe nucleic acid amplification methods
First Claim
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1. A method of targeted amplification of nucleic acids comprising:
- contacting a sample containing one or more target nucleic acid sequences with one or more precircle probes, wherein each precircle probe comprises at least a first and second targeting domain which hybridize to a target nucleic acid sequence and which flank at least one universal priming sequence, under conditions appropriate for hybridization of the first and second targeting domains of a precircle probe with a target nucleic acid sequence;
extending the precircle probe with a polymerase such that the target nucleic acid sequence is copied, thereby producing an extended precircle probe;
ligating one end of the extended precircle probe to the other end to create a closed circular probe;
optionally removing linear nucleic acid sequences remaining in the sample;
cleaving the closed circular probe prior to amplification; and
amplifying the cleaved circular probe to produce amplified target nucleic acid sequence.
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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.
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Citations
6 Claims
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1. A method of targeted amplification of nucleic acids comprising:
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contacting a sample containing one or more target nucleic acid sequences with one or more precircle probes, wherein each precircle probe comprises at least a first and second targeting domain which hybridize to a target nucleic acid sequence and which flank at least one universal priming sequence, under conditions appropriate for hybridization of the first and second targeting domains of a precircle probe with a target nucleic acid sequence;
extending the precircle probe with a polymerase such that the target nucleic acid sequence is copied, thereby producing an extended precircle probe;
ligating one end of the extended precircle probe to the other end to create a closed circular probe;optionally removing linear nucleic acid sequences remaining in the sample; cleaving the closed circular probe prior to amplification; and amplifying the cleaved circular probe to produce amplified target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6)
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Current AssigneeBoard of Trustees of the Leland Stanford Junior University (Stanford University)
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Original AssigneeBoard of Trustees of the Leland Stanford Junior University (Stanford University)
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InventorsJain, Maneesh, Ronaghi, Mostafa, Stolc, Viktor, Willis, Thomas D., Hardenbol, Paul, Davis, Ronald W.
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Primary Examiner(s)Staples; Mark
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Application NumberUS12/709,319Publication NumberTime in Patent Office536 DaysField of SearchNoneUS Class Current435/91.1CPC Class CodesC12Q 1/6827 for detection of mutation o...C12Q 1/6858 Allele-specific amplificationC12Q 1/686 Polymerase chain reaction [...C12Q 2525/307 Circular oligonucleotidesC12Q 2531/113 PCRC12Q 2537/143 Multiplexing, i.e. use of m...
