Direct multiplex characterization of genomic DNA
OwnerThomas Willis, Paul Hardenbol +4inventors
InventorsThomas Willis, Paul Hardenbol, Maneesh Jain, Viktor Stolc, Mostafa Ronaghi, Ronald Davis
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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.
Claims26
1-47. (canceled)
A method of determining the identity of a nucleotide at a detection position in a target sequence comprising a first domain and a second domain, said method comprising the steps of:
- a) hybridizing said first domain to a first end of a padlock probe wherein said padlock probe comprises:
- i) a first end that is complementary to said first domain of said target sequence;
- ii) a barcode sequence;
- iii) a second end that is complementary to said second domain of said target sequence wherein said first end of said padlock probe and said second end of said padlock probe are located at the ends of said padlock probe;
- and
- iv) two priming sites
- b) hybridizing said second domain of said target sequence to said second end of said padlock probe;
- c) covalently attaching said first end of said padlock probe and said second end of said padlock probe while hybridized to said target sequence;
- d) amplifying a sequence comprising at least a portion of said padlock probe following said covalently attaching, thereby producing amplification products comprising said barcode sequence;
- and
- e) binding said barcode sequence of said amplification product to a substrate wherein said substrate comprises an attached barcode capture probe and/or an attached complement of said barcode sequence;
- thereby identifying said nucleotide at said detection position.
A method of determining the identity of a nucleotide at a detection position in a target sequence comprising a first domain and a second domain, said method comprising the steps of:
- a) hybridizing said first domain to a first end of a padlock probe wherein said padlock probe comprises:
- i) a first end that is complementary to said first domain of said target sequence;
- ii) a barcode sequence;
- iii) a cleavage site;
- iv) a second end that is complementary to said second domain of said target sequence wherein said first end of said padlock probe and said second end of said padlock probe are located at the ends of said padlock probe;
- and
- v) two priming sites;
- b) hybridizing said second domain of said target sequence to said second end of said padlock probe;
- c) covalently attaching said first end of said padlock probe and said second end of said padlock probe while hybridized to said target sequence;
- d) cleaving said covalently attached padlock probe at said cleavage site;
- and
- e) binding said barcode sequence of said covalently attached padlock probe to a substrate wherein said substrate comprises a capture probe and/or an attached complement of said adapter sequence;
- thereby identifying said nucleotide at said detection position.
Dependencies are inferred from the claim text.