Methods for controlling amplification

Abstract

Methods of amplifying nucleic acid on a solid support are described. Beads and template, each in known concentrations, are employed so a range of template to bead ratios can be exploited. Where the beads contain primers, the template can be amplified. After amplification, non-covalently bound template is removed, so as to leave beads with extended primers (or beads with primers that were not extended).

1 . A method of amplifying nucleic acid on a solid support, comprising:
 (a) providing i) a population of beads, each bead comprising one or more amplification primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of nucleic acid template molecules,   (b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture;   (c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;   (d) manipulating said treated beads so as to remove at least a portion of said bound template so as to create manipulated beads; and   (e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein said manipulating of step d) comprises washing said treated beads with a denaturing solution. 
     
     
         3 . The method of  claim 2 , wherein said denaturing solution comprises NaOH. 
     
     
         4 . The method of  claim 3 , wherein prior to step d) between 1 and 10 primers per bead are extended. 
     
     
         5 . The method of  claim 3 , wherein prior to step d) some beads comprise no extended primers. 
     
     
         6 . The method of  claim 1 , wherein at step a) a known concentration of beads is provided. 
     
     
         7 . The method of  claim 6 , wherein at step a) a known concentration of nucleic acid template molecules is provided. 
     
     
         8 . The method of  claim 7 , wherein at step b) the number of template molecules to beads is less than one. 
     
     
         9 . The method of  claim 8 , wherein at step c) fewer than 50% of the beads comprise non-covalently bound template. 
     
     
         10 . The method of  claim 1 , wherein the amplification primers comprise a sequence which provides a code. 
     
     
         11 . The method of  claim 10 , wherein said code identifies the origin of the nucleic acid templates. 
     
     
         12 . The method of  claim 11 , wherein the origin of the nucleic acid template is a patient and the code identifies the patient. 
     
     
         13 . The method of  claim 10 , wherein said code identifies the bead. 
     
     
         14 . The method of  claim 1 , wherein each bead of step (a) comprises a forward and a reverse PCR primer. 
     
     
         15 . A method of amplifying nucleic acid on a solid support, comprising:
 a) providing i) a population of beads, each bead comprising forward and reverse PCR primers primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of nucleic acid template molecules,   b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture;   c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;   d) washing said treated beads with a denaturing solution so as to create manipulated beads; and   e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.   
     
     
         16 . The method of  claim 15 , further comprising:
 (f) treating said immobilized amplified nucleic acid so as to release at least a portion from said loaded beads so as to create free amplified nucleic acid.   
     
     
         17 . The method of  claim 15 , further comprising:
 (f) transferring at least a portion of said immobilized amplified nucleic acid to a non-bead solid support.   
     
     
         18 . The method of  claim 15 , wherein prior to step (a) said forward and reverse PCR primers comprised 5′ amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups. 
     
     
         19 . The method of  claim 15 , wherein said forward and reverse PCR primers have a region that is completely complementary to a portion of the APC gene segment 3. 
     
     
         20 . The method of  claim 15 , wherein said forward primer comprises a portion encoding an N-terminal epitope tag and said reverse primer comprises a portion encoding a C-terminal epitope tag. 
     
     
         21 . The method of  claim 15 , wherein said mixture is created under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 0.1:1 and 2:1. 
     
     
         22 . The method of  claim 15 , wherein said mixture is created under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 2:1 and 500, 000:1. 
     
     
         23 . The method of  claim 22 , wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 1000:1 and 100, 000:1. 
     
     
         24 . The method of  claim 22 , wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 10,000:1 and 100, 000:1 
     
     
         25 . The method of  claim 22 , wherein ratio of the number of nucleic acid template molecules to the number of beads is between 1000:1 and 10,000:1. 
     
     
         26 . The method of  claim 15 , wherein the percentage of unloaded beads is between approximately 50% and 95%, as measured by fluorescence. 
     
     
         27 . The method of  claim 15 , wherein the percentage of loaded beads is between approximately 1% and 5%, as measured by fluorescence. 
     
     
         28 . The method of  claim 15 , wherein the percentage of loaded beads is between approximately 5% and 50%, as measured by fluorescence. 
     
     
         29 . A method of amplifying nucleic acid on a solid support, comprising: a) providing i) a population of a known concentration of beads, each bead comprising one or more amplification primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of a known concentration of nucleic acid template molecules; b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 1:1 and 10,000:1; c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads; d) manipulating said treated beads so as to remove at least a portion of said bound template so as to create manipulated beads; and e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid. 
     
     
         30 . The method of  claim 29 , wherein each bead of step (a) comprises a forward and a reverse PCR primer. 
     
     
         31 . The method of  claim 29 , wherein said manipulating comprises washing said treated beads with a denaturing solution 
     
     
         32 . The method of  claim 31 , wherein said denaturing solution comprises NaOH. 
     
     
         33 . The method of  claim 29 , wherein said washing removes the majority of said non-covalently bound template. 
     
     
         34 . The method of  claim 29 , further comprising:
 (f) treating said immobilized amplified nucleic acid so as to release at least a portion from said loaded beads so as to create free amplified nucleic acid.   
     
     
         35 . The method of  claim 29 , further comprising:
 (f) transferring at least a portion of said immobilized amplified nucleic acid to a non-bead solid support.   
     
     
         36 . The method of  claim 30 , wherein prior to step (a) said forward and reverse PCR primers comprised 5′ amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups. 
     
     
         37 . The method of  claim 30 , wherein said forward and reverse PCR primers have a region that is completely complementary to a portion of the APC gene segment 3. 
     
     
         38 . The method of  claim 30 , wherein said forward primer comprises a portion encoding an N-terminal epitope tag and said reverse primer comprises a portion encoding a C-terminal epitope tag. 
     
     
         39 . The method of  claim 29 , wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 1:1 and 10:1. 
     
     
         40 . The method of  claim 29 , wherein the ratio of the number of nucleic acid template molecules to the number of beads is between 10:1 and 100:1 
     
     
         41 . The method of  claim 29 , wherein ratio of the number of nucleic acid template molecules to the number of beads is between 100:1 and 1,000:1. 
     
     
         42 . The method of  claim 29  wherein the percentage of unloaded beads is between approximately 50% and 95%, as measured by fluorescence. 
     
     
         43 . The method of  claim 29  wherein the percentage of loaded beads is between approximately 1% and 5%, as measured by fluorescence. 
     
     
         44 . The method of  claim 29  wherein the percentage of loaded beads is between approximately 5% and 50%, as measured by fluorescence. 
     
     
         45 . A method of amplifying nucleic acid on a solid support, comprising:
 a) providing i) a population of a known concentration of beads, each bead comprising one or more amplification primers, ii) a solution of amplification reagents comprising a thermostable polymerase, and iii) a population of a known concentration of nucleic acid template molecules,   b) mixing said beads and said template molecules in a first aliquot of said solution of amplification reagents so as to create a mixture under the conditions such that the ratio of the number of nucleic acid template molecules to the number of beads is between 0.1:1 and 2:1;   c) treating the mixture under conditions such that at least a portion of said template molecules non-covalently bind to at least a portion of said beads to create bound template, and at least a portion of said primers on at least a portion of said beads are extended by said polymerase, so as to create treated beads;   d) exposing said treated beads to a denaturing solution so as to create manipulated beads; and   e) contacting said manipulated beads with a second aliquot of said solution of amplification reagents under conditions such that at least a portion of said extended primers is amplified to create loaded beads comprising immobilized amplified nucleic acid and unloaded beads lacking amplified nucleic acid.   
     
     
         46 . The method of  claim 45 , wherein each bead of step (a) comprises a forward and a reverse PCR primer. 
     
     
         47 . The method of  claim 45 , wherein said denaturing solution comprises NaOH and said exposing comprises at least two washings of the beads. 
     
     
         48 . The method of  claim 47 , wherein said washings remove at least a portion of said non-covalently bound template. 
     
     
         49 . The method of  claim 47 , wherein said washings removes the majority of said non-covalently bound template. 
     
     
         50 . The method of  claim 45 , further comprising:
 (f) treating said immobilized amplified nucleic acid so as to release at least a portion from said loaded beads so as to create free amplified nucleic acid.   
     
     
         51 . The method of  claim 45 , further comprising:
 (f) transferring at least a portion of said immobilized amplified nucleic acid to a non-bead solid support.   
     
     
         52 . The method of  claim 45  wherein the percentage of unloaded beads is between approximately 50% and 99%, as measured by fluorescence. 
     
     
         53 . The method of  claim 45  wherein the percentage of loaded beads is between approximately 0.1% and 2%, as measured by fluorescence.