Method of nucleic acid amplification

Abstract

A nucleic acid molecule can be annealed to an appropriate immobilized primer. The primer can then be extended and the molecule and the primer can be separated from one another. The extended primer can then be annealed to another immobilized primer and the other primer can be extended. Both extended primers can then be separated from one another and can be used to provide further extended primers. The process can be repeated to provide amplified, immobilized nucleic acid molecules. These can be used for many different purposes, including sequencing, screening, diagnosis, in situ nucleic acid synthesis, monitoring gene expression, nucleic acid fingerprinting, etc.

1 . (canceled) 
     
     
         2 . 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 extending the primers while the primers are hybridized to the replicated and solid support bound fragments.   
     
     
         3 . The process of  claim 2  wherein steps b and c are repeated at least once prior to binding to the solid support in step d. 
     
     
         4 . 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 molecules replicated from said nucleic acid sample, with nucleic acid molecules comprising sequence-specific binding activity under conditions which permit binding;   b. 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 linked;   c. replicating the linked molecules of step b;   d. binding the replicated molecules of step c to a solid support to form bound replicated molecules;   e. identifying nucleic acid sequences within said replicated molecules.   
     
     
         5 . The process of  claim 4  wherein linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         6 . The process of  claim 4  wherein linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         7 . The process of  claim 4  whereby the replicating in step c includes amplification with a molecule with polymerase activity. 
     
     
         8 . The process of  claim 4  wherein the bound replicated molecules of step d, are amplified on the solid support by contacting them with a molecule with polymerase activity. 
     
     
         9 . The process of  claim 4  whereby the linked molecules comprising sequence-specific binding activity of step b are replicated in step c by contacting said linked molecules with primers with adapter sequences. 
     
     
         10 . The process of  claim 9  wherein the molecules replicated by primers with adapter sequences are bound to the solid support by hybridizing said adapter sequences. 
     
     
         11 . 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 molecules replicated from said nucleic acid sample, with nucleic acid molecules comprising sequence-specific binding activity under conditions which permit binding;   b. 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 linked;   c. binding the linked molecules to a solid support and replicating said bound molecules;   d. identifying nucleic acid sequences within said replicated molecules.   
     
     
         12 . The process of  claim 11  wherein linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         13 . The process of  claim 11  wherein linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         14 . The process of  claim 11  whereby the replicating in step c includes amplification with a molecule with polymerase activity. 
     
     
         15 . The process of  claim 11  wherein the bound linked molecules of step c, are replicated on the solid support by contacting them with a molecule with polymerase activity. 
     
     
         16 . The process of  claim 11  whereby the linked molecules comprising sequence-specific binding activity of step b are replicated in step c by contacting said linked molecules with primers with adapter sequences before binding said molecules to the solid support. 
     
     
         17 . The process of  claim 16  wherein the molecules replicated by contacting them with primers with adapter sequences are bound to the solid support by hybridizing said adapter sequences. 
     
     
         18 . The process of  claim 4 , wherein said identification comprises identifying nucleic acid sequences within bound and replicated molecules. 
     
     
         19 . The process of  claim 18  wherein linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         20 . The process of  claim 18  wherein linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         21 . The process of  claim 18  whereby the replicating in step c includes amplification with a molecule with polymerase activity. 
     
     
         22 . The process of  claim 18  wherein the bound replicated molecules of step d, are amplified on the solid support by contacting them with a molecule with polymerase activity. 
     
     
         23 . The process of  claim 18  whereby the linked molecules comprising sequence-specific binding activity of step b are replicated in step c by contacting said linked molecules with primers with adapter sequences. 
     
     
         24 . The process of  claim 23  wherein the molecules replicated by primers with adapter sequences are bound to the solid support by hybridizing said adapter sequences. 
     
     
         25 . The process of  claim 11 , wherein said identification comprises identifying nucleic acid sequences within bound and replicated molecules. 
     
     
         26 . The process of  claim 25  wherein linking said molecules comprising sequence-specific binding activity in step b, is achieved by ligating said molecules. 
     
     
         27 . The process of  claim 25  wherein linking said molecules comprising sequence-specific binding activity in step b is achieved by modifying one or more said molecules with a polymerase. 
     
     
         28 . The process of  claim 25  whereby the replicating in step c includes amplification with a molecule with polymerase activity. 
     
     
         29 . The process of  claim 25  wherein the bound linked molecules of step c, are replicated on the solid support by contacting them with amplification with a molecule with polymerase activity. 
     
     
         30 . The process of  claim 25  whereby the linked molecules comprising sequence-specific binding activity of step b are replicated in step c by contacting said linked molecules with primers with adapter sequences before binding said molecules to the solid support. 
     
     
         31 . The process of  claim 30  wherein the molecules replicated with primers with adapter sequences are bound to the solid support by hybridizing said adapter sequences.