Single molecule sequencing of captured nucleic acids
View Patent ↗The invention provides methods and devices for detecting, enumerating or identifying target nucleic acid molecules using immobilized capture probes and single molecule sequencing techniques.
1. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:
capturing target nucleic acids with a sequence-specific capture probe to produce a target/probe duplex;
melting said target/probe duplex to release said target nucleic acids;
annealing a primer to the target nucleic acids to produce a target/primer duplex;
attaching said target/primer duplex to a surface such that at least a portion of said target/primer duplex is individually optically resolvable;
introducing a polymerase and at least one nucleotide species comprising an optically-detectable label under conditions sufficient for template-dependent nucleotide addition;
removing unincorporated nucleotide; and
identifying nucleotide species incorporated into the extended primer, thereby obtaining sequence information from said target.
2. The method of claim 1 , further comprising the step of removing said optically-detectable label from nucleotide incorporated into said primer.
3. The method of claim 1 , wherein said capturing step comprises hybridizing said target nucleic acids to capture probes immobilized in a polyacrylamide gel.
4. The method of claim 1 , wherein said capture probes are attached to a bead.
5. The method of claim 1 , wherein said optically-detectable label is a fluorescent label.
6. The method of claim 1 , wherein said introducing, removing, and identifying steps are repeated at least once.
7. The method of claim 1 , wherein said surface comprises an epoxide.
8. The method of claim 1 , wherein said attaching step comprises modifying said target nucleic acid to introduce a 3′ or 5′ amine.
9. The method of claim 1 , wherein said surface is streptavidnated.
10. The method of claim 9 , wherein said target and/or said primer comprise a biotin end label.
11. The method of claim 1 , wherein the sequence specific-capture probes of multiple sequences are used.
12. The method of claim 1 , wherein the target nucleic acids are produced as a result of fragmentation by sonication.