Methods of enriching for and identifying polymorphisms

Abstract

The invention encompasses methods for enriching for and identifying a polymorphism within a nucleic acid sample either by separating a subset of a nucleic acid sample or by selectively replicating a subset of a nucleic acid sample such that the polymorphism is contained within a nucleic acid population with reduced complexity, and then identifying the polymorphism within the enriched nucleic acid sample. Methods also are disclosed for enriching for and identifying a polymorphism by contacting a nucleic acid sample that includes a subset of nucleic acid molecules having a sequence that binds to a sequence-specific binding activity with a molecule having a sequence-specific binding activity under conditions which permit specific binding, such that the subset of nucleic acid molecules bound to the activity is enriched for nucleic acid molecules having the sequence recognized by the sequence-specific binding activity, and detecting a polymorphism with respect to a reference sequence in the subset of nucleic acid molecules.

1 - 35 . (canceled) 
     
     
         36 . A process that can be used to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to a reference sequence, the process comprising:
 a. fragmenting nucleic acids in said sample;   b. ligating adapter sequences to the nucleic acid fragments generated in step a;   c. replicating said adapter linked nucleic acid fragments of step b;   d. binding said replicated and adapter ligated nucleic acid fragments to a solid support by hybridizing oligonucleotides attached to the solid support to the adapter sequences; and   e. identifying nucleic acid sequences within said replicated and solid support bound nucleic acid fragments by hybridizing primers to the bound and replicated fragments and ligating the primers to oligonucleotides which are also hybridized to the replicated and solid support bound fragments.   
     
     
         37 . The process of claim  1  wherein steps b and c are repeated at least once prior to binding to the solid support in step d. 
     
     
         38 . A process to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to one or more reference sequences, the process comprising:
 a. contacting a nucleic acid sample, or a replica thereof, with molecules comprising sequence-specific binding activity under conditions which permit binding;   b. operatively linking said molecules comprising sequence-specific binding activity to one another when said molecules bind to the nucleic acid sample, or a replica thereof, under conditions which permit said molecules to be operatively linked;   c. replicating the operatively linked molecules of step b;   d. binding the replicated molecules of step c to a solid support to form bound replicated molecules; and   e. identifying nucleic acid sequences within said replicated molecules.   
     
     
         39 . A process of  claim 38  wherein operatively linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         40 . A process of  claim 38  wherein operatively linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         41 . A process of  claim 38  whereby the replicating in step c includes amplification using a molecule with polymerase activity. 
     
     
         42 . A process of  claim 38  wherein the bound replicated molecules of step d, are amplified on the solid support using a molecule with polymerase activity. 
     
     
         43 . A process of  claim 38  whereby the operatively linked molecules comprising sequence-specific binding activity of step b are replicated in step c using primers with adapter sequences. 
     
     
         44 . A process of  claim 43  wherein the molecules replicated using primers with adapter sequences are bound to the solid support using said adapter sequences. 
     
     
         45 . A process to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to one or more reference sequences, the process comprising:
 a. contacting a nucleic acid sample, or a replica thereof, with molecules comprising sequence-specific binding activity under conditions which permit binding;   b. operatively linking said molecules comprising sequence-specific binding activity to one another when said molecules bind to the nucleic acid sample, or a replica thereof, under conditions which permit said molecules to be operatively linked;   c. binding the operatively linked molecules to a solid support and replicating said bound molecules; and   d. identifying nucleic acid sequences within said replicated molecules.   
     
     
         46 . A process of  claim 45  wherein operatively linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         47 . A process of  claim 45  wherein operatively linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         48 . A process of  claim 45  whereby the replicating in step c includes amplification using a molecule with polymerase activity. 
     
     
         49 . A process of  claim 45  wherein the bound operatively linked molecules of step c, are replicated on the solid support using amplification with a molecule with polymerase activity. 
     
     
         50 . A process of  claim 45  whereby the operatively linked molecules comprising sequence-specific binding activity of step b are replicated in step c using primers with adapter sequences before binding said molecules to the solid support. 
     
     
         51 . A process of  claim 50  wherein the molecules replicated using primers with adapter sequences are bound to the solid support using said adapter sequences. 
     
     
         52 . A process to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to one or more reference sequences, the process comprising:
 a. contacting a nucleic acid sample, or a replica thereof, with molecules comprising sequence-specific binding activity under conditions which permit binding;   b. operatively linking said molecules comprising sequence-specific binding activity to one another when said molecules bind to the nucleic acid sample, or a replica thereof, under conditions which permit said molecules to be operatively linked;   c. replicating the operatively linked molecules of step b;   d. binding the replicated molecules of step c to a solid support to form bound replicated molecules; and   e. identifying nucleic acid sequences within said bound and replicated molecules.   
     
     
         53 . A process of  claim 52  wherein operatively linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         54 . A process of  claim 52  wherein operatively linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         55 . A process of  claim 52  whereby the replicating in step c includes amplification using a molecule with polymerase activity. 
     
     
         56 . A process of  claim 52  wherein the bound replicated molecules of step d, are amplified on the solid support using a molecule with polymerase activity. 
     
     
         57 . A process of  claim 52  whereby the operatively linked molecules comprising sequence-specific binding activity of step b are replicated in step c using primers with adapter sequences. 
     
     
         58 . A process of  claim 57  wherein the molecules replicated using primers with adapter sequences are bound to the solid support using said adapter sequences. 
     
     
         59 . A process to identify in a nucleic acid sample the presence or absence of nucleic acid sequence differences, wherein each said difference is with respect to one or more reference sequences, the process comprising:
 a. contacting a nucleic acid sample, or a replica thereof, with molecules comprising sequence-specific binding activity under conditions which permit binding;   b. operatively linking said molecules comprising sequence-specific binding activity to one another when said molecules bind to the nucleic acid sample, or a replica thereof, under conditions which permit said molecules to be operatively linked;   c. binding the operatively linked molecules to a solid support and replicating said bound molecules; and   d. identifying nucleic acid sequences within said bound and replicated molecules.   
     
     
         60 . A process of  claim 59  wherein operatively linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         61 . A process of  claim 59  wherein operatively linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         62 . A process of  claim 59  whereby the replicating in step c includes amplification using a molecule with polymerase activity. 
     
     
         63 . A process of  claim 59  wherein the bound operatively linked molecules of step c, are replicated on the solid support using amplification with a molecule with polymerase activity. 
     
     
         64 . A process of  claim 59  whereby the operatively linked molecules comprising sequence-specific binding activity of step b are replicated in step c using primers with adapter sequences before binding said molecules to the solid support. 
     
     
         65 . A process of  claim 64  wherein the molecules replicated using primers with adapter sequences are bound to the solid support using said adapter sequences.