Methods and systems for solution based sequence enrichment

Abstract

The present invention provides methods and systems for the capture and enrichment of target nucleic acids and analysis of the enriched target nucleic acids. In particular, the present invention provides for the enrichment of targeted sequences in a solution based format.

1 . A method for isolating and reducing the complexity of a plurality of nucleic acid sequences comprising:
 a) providing:
 i) a solid support wherein said solid support comprises hybridization probes hybridizable to target nucleic acid sequences, 
 ii) a fragmented nucleic acid sample comprising target nucleic acid sequences, 
   b) amplifying said hybridization probes wherein the amplification products comprise a binding moiety and wherein said amplification products are maintained in solution,   c) hybridizing said nucleic acid sample to said amplification products in solution such that hybridization between said amplification products and target nucleic acid sequences is allowed to occur,   d) separating the target nucleic acid/amplification product hybridization complexes from non-specifically hybridized nucleic acids by said binding moiety, and   e) eluting the hybridized target nucleic acid sequences from the complex thereby isolating and reducing the complexity of a plurality of nucleic acid sequences.   
     
     
         2 . The method of  claim 1 , wherein said solid support is a microarray slide. 
     
     
         3 . The method of  claim 1 , wherein said nucleic acid sample is a fragmented genomic DNA sample. 
     
     
         4 . The method of  claim 1 , wherein said fragmented genomic DNA sample further comprises adaptor molecules at one or both ends of the fragments of genomic DNA sample. 
     
     
         5 . The method of  claim 1 , wherein said hybridization probes further comprise primer binding sequences at one or both ends of said probes. 
     
     
         6 . The method of  claim 5 , wherein said primer binding sequences when present at both ends of the probes are the same. 
     
     
         7 . The method of  claim 5 , wherein said primer binding sequences when present at both ends of the probes are different. 
     
     
         8 . The method of  claim 1 , wherein said amplifying comprises exponential polymerase chain reaction. 
     
     
         9 . The method of  claim 8 , wherein said amplifying further comprises asymmetric polymerase chain reaction. 
     
     
         10 . The method of  claim 1 , wherein said binding moiety is a biotin binding moiety. 
     
     
         11 . The method of  claim 10 , wherein said separating comprises binding said biotin binding moiety to a streptavidin coated substrate. 
     
     
         12 . The method of  claim 11 , wherein said streptavidin coated substrate is a streptavidin coated paramagnetic particle. 
     
     
         13 . The method of  claim 1 , further comprising washing said separated target nucleic acid/amplification product hybridization complexes prior to elution. 
     
     
         14 . The method of  claim 1 , further comprising sequencing the eluted target nucleic acid sequences. 
     
     
         15 . A method for isolating and reducing the complexity of a plurality of nucleic acid sequences comprising:
 a) providing:
 i) a solid support wherein said solid support comprises hybridization probes hybridizable to target nucleic acid sequences, 
 ii) a nucleic acid sample comprising target nucleic acid sequences, 
   b) amplifying said hybridization probes wherein the amplification products comprise a binding moiety and wherein said amplification products are maintained in solution,   c) hybridizing said nucleic acid sample to said amplification products in solution such that hybridization between said amplification products and target nucleic acids is allowed to occur,   d) separating the target nucleic acid/amplification product hybridization complexes from non-specifically hybridized nucleic acids by said binding moiety,   e) eluting the hybridized target nucleic acid sequences from the complexes thereby isolating and reducing the complexity of a plurality of nucleic acid sequences, and   f) sequencing the eluted target nucleic acid sequences.   
     
     
         16 . The method of  claim 15 , wherein said solid support is a microarray slide. 
     
     
         17 . The method of  claim 15 , wherein said nucleic acid sample is a fragmented genomic DNA sample. 
     
     
         18 . The method of  claim 15 , wherein said fragmented genomic DNA sample further comprises adaptor molecules at one or both ends of the fragments of genomic DNA sample. 
     
     
         19 . The method of  claim 15 , wherein said hybridization probes further comprise primer binding sequences at one or both ends of said probes. 
     
     
         20 . The method of  claim 19 , wherein said primer binding sequences when present at both ends of the probes are the same. 
     
     
         21 . The method of  claim 19 , wherein said primer binding sequences when present at both ends of the probes are different. 
     
     
         22 . The method of  claim 15 , wherein said amplifying comprises exponential polymerase chain reaction. 
     
     
         23 . The method of  claim 22 , wherein said amplifying further comprises asymmetric polymerase chain reaction. 
     
     
         24 . The method of  claim 15 , wherein said binding moiety is a biotin binding moiety. 
     
     
         25 . The method of  claim 24 , wherein said separating comprises binding said biotin binding moiety to a streptavidin coated substrate. 
     
     
         26 . The method of  claim 25 , wherein said streptavidin coated substrate is a streptavidin coated paramagnetic particle. 
     
     
         27 . The method of  claim 15 , further comprises washing said separated target nucleic acid/amplification product hybridization complex prior to elution. 
     
     
         28 . A kit comprising:
 a) hybridization probe sequences comprising a binding moiety wherein said probe sequences are designed to hybridize to one or more target nucleic acid sequences and wherein said probe sequences are in solution,   b) a substrate comprising a binding partner for binding said binding moiety, and   c) instructions for performing the methods of  claim 1 .   
     
     
         29 . The kit of  claim 28 , further comprising one or more of a hybridization solution, a wash solution and an elution solution. 
     
     
         30 . The kit of  claim 29 , further comprising a magnet.