Solid phase methods for amplifying multiple nucleic acids

Abstract

A multi-stage bridge amplification method which uses a recovered single-stranded amplification single-stranded nucleic acid molecule to initiate second and subsequent stages of bridge amplification is described.

What is claimed is:  
     
         1 . A solid-phase, multi-stage method of amplifying one, or more, target nucleic acid molecules comprising two or more stages of bridge amplification, wherein single-stranded amplification molecules produced in the first stage of bridge amplification initiate a second stage of bridge amplification, and each subsequent stage of bridge amplification is initiated with single-stranded amplification molecules produced in the previous stage of bridge amplification.  
     
     
         2 . The method of  claim 1 , wherein each stage of bridge amplification comprises the steps of: 
 (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support, and    (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers by the formation of bridge amplification double-stranded molecules.    
     
     
         3 . The method of  claim 2 , wherein the thermocycling step (b) comprises about 5 to about 50 thermocycles.  
     
     
         4 . The method of  claim 2  further comprising the steps of: 
 (c) cleaving and denaturing the bridge amplification double-stranded molecules to form single-stranded amplification molecules;  
 (d) recovering the single-stranded amplification molecules of step (c), and  
 (e) contacting the single-stranded amplification molecules with a fresh solid support, and  
 (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules.  
 
     
     
         5 . A method of amplifying one, or more, target nucleic acid molecules using a solid support comprising two or more immobilized oligonucleotide primers, of which at least one primer specifically hybridizes to the target nucleic acid molecules, comprising the steps of: 
 (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support;    (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers forming a double-stranded amplification molecule;    (c) cleaving and denaturing the double-stranded amplification molecule to form single-stranded amplification molecules;    (d) recovering the single-stranded amplification molecules of step (c),    (e) contacting the single-stranded amplification molecules with a fresh solid support, and    (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules.    
     
     
         6 . The method of  claim 5 , wherein the thermocycling step (b) comprises about 5 to about 50 thermocycles.  
     
     
         7 . The method of  claim 6 , wherein each thermocycle comprises from about five seconds to about one minute each at 95° C., 60° C. and 72° C.  
     
     
         8 . A method of amplifying one, or more, target nucleic acid molecules using a solid support comprising two or more immobilized oligonucleotide primers, of which at least one specifically hybridizes to the target nucleic acid molecule, comprising the steps of: 
 (a) forming a hybridization complex comprising a single-stranded target nucleic acid molecule hybridized to a first oligonucleotide primer immobilized to the solid support by contacting the target nucleic acid molecule with the solid support, under conditions suitable for hybridization;    (b) forming a first double-stranded amplification molecule comprising the target nucleic acid molecule hybridized to a first single-stranded amplification molecule by contacting the hybridization complex of step (a) with amplification reagents under conditions suitable for a primer-mediated polymerase extension reaction, wherein a first single-stranded amplification molecule is formed by extending the first primer with deoxynucleotides to form a complementary strand to the target nucleic acid molecule;    (c) denaturing the first double-stranded amplification molecule of (b), thereby releasing the single-stranded target molecule from the first double-stranded amplification molecule;    (d) forming a first bridge hybridization complex comprising the first single-stranded amplification molecule of step (b) and a second oligonucleotide primer immobilized to the solid support, under conditions suitable for hybridization;    (e) forming a second double-stranded amplification molecule by contacting the bridge hybridization complex of step (d) with amplification reagents, under conditions suitable for a primer-mediated amplification reaction, wherein a second single-stranded amplification molecule is formed that is complementary to and base-paired with the first single-stranded amplification molecule of step (d);    (f) denaturing the second double-stranded amplification molecule of (e) to produce first and second single-stranded amplification molecules immobilized to the solid support;    (g) forming second bridge hybridization complexes comprising 
 (i) the first single-stranded amplification molecule of step (f) and a second oligonucleotide primer immobilized to the solid support, under conditions suitable for hybridization, and  
 (ii) the second single-stranded amplification molecule of step (f) and a first oligonucleotide primer immobilized to the solid support, under conditions suitable for hybridization;  
   (h) forming a third and fourth double-stranded amplification molecule by contacting the second bridge hybridization complexes of step (g) with amplification reagents, under conditions suitable for a primer-mediated amplification reaction, wherein 
 (i) the third double-stranded amplification molecule comprises a nascent third single-stranded amplification molecule that is complementary to and is base-paired with the first single-stranded amplification molecule, and  
 (ii) the fourth double-stranded amplification molecule comprises a nascent fourth single-stranded amplification molecule that is complementary to and is base-paired with the second single-stranded amplification molecule;  
   (i) cleaving one, or more, first oligonucleotide primers, thereby cleaving the third and fourth double-stranded amplification molecules from at least one attachment to the solid support, or alternatively cleaving one, or more, second oligonucleotide primers, thereby cleaving the third and fourth double-stranded amplification molecules from at least one attachment to the solid support;    (j) denaturing the cleaved amplification double-stranded molecules of (h), thereby releasing amplification single-stranded molecules from the solid support;    (k) applying released single-stranded molecules from step (j) to fresh solid supports comprising unused immobilized oligonucleotide primers, and    (l) repeating steps (a) through (k) one, or more, times, thereby amplifying the target nucleic acid molecules.    
     
     
         9 . The method of  claim 8 , wherein steps (f), (g) and (h) are repeated from about 5 to about 50 times.  
     
     
         10 . The method of  claim 8 , wherein the oligonucleotide primers are immobilized to the solid support through one, or more, covalent interactions.  
     
     
         11 . The method of  claim 10 , wherein the oligonucleotide primers are immobilized via co-polymerization with a polymeric surface layer on the solid support.  
     
     
         12 . The method of  claim 8  wherein in step (i), the oligonucleotide primers are cleaved using chemical means.  
     
     
         13 . The method of  claim 12 , wherein the chemical means is selected from reagents that reduce disulfide bonds.  
     
     
         14 . The method of  claim 13 , wherein the reducing reagents are selected from the group consisting of: DTT, β-mercaptoethanol and TCEP.  
     
     
         15 . The method of  claim 8  wherein in step (i), the oligonucleotide primers are cleaved using photochemical means.  
     
     
         16 . The method of  claim 15 , wherein the oligonucleotide primers comprise a nitrophenol moiety.  
     
     
         17 . The method of  claim 8  wherein in step (i), the oligonucleotide primers are cleaved using enzymatic means.  
     
     
         18 . The method of  claim 17 , wherein the enzymatic means is accomplished using one, or more, restriction endonucleases.  
     
     
         19 . The method of  claim 8 , wherein the denaturant used for denaturing the double-stranded amplification molecules is denatured using at least one of the following denaturants selected from the group consisting of: high temperature, high pH, organic solvent, chaotropic agent and combinations thereof.  
     
     
         20 . The method of  claim 8 , wherein the material composition of the solid support is selected from the group consisting of: plastic, glass, silica, nylon, metal, metal alloys, polyacrylamide, polyacrylates, crosslinked-dextran and combinations thereof.  
     
     
         21 . The method of  claim 8 , wherein the solid support is a bead.  
     
     
         22 . The method of  claim 21 , wherein the bead comprises one, or more, oligonucleotide primer sets for more than one target nucleic acid molecule.  
     
     
         23 . The method of  claim 8 , wherein the oligonucleotide primers are from about 5 to about 500 in nucleotide length.  
     
     
         24 . The method of  claim 8 , wherein one or more amplification products are labeled.  
     
     
         25 . The method of  claim 24 , wherein the label is selected from the group consisting of: radioactivity, chemiluminescence, luminescence and fluorescence.  
     
     
         26 . A solid-phase, multi-stage method of detecting the presence or absence of one, or more, target nucleic acid molecules comprising two or more stages of bridge amplification, wherein single-stranded amplification molecules produced in the first stage of bridge amplification initiate a second stage of bridge amplification, and each subsequent stage of bridge amplification is initiated with single-stranded amplification molecules produced in the previous stage of bridge amplification.  
     
     
         27 . The method of  claim 26 , wherein one or more single-stranded amplification molecules are labeled.  
     
     
         28 . The method of  claim 27 , wherein the label is selected from the group consisting of: radioactivity, chemiluminescence, luminescence and fluorescence.  
     
     
         29 . The method of  claim 26 , wherein each stage of bridge amplification comprises the steps of: 
 (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support, and    (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers by the formation of bridge amplification double-stranded molecules.    
     
     
         30 . The method of  claim 29 , wherein the thermocycling step (b) comprises about 5 to about 50 thermocycles.  
     
     
         31 . The method of  claim 26  further comprising the steps of: 
 (c) cleaving and denaturing the bridge amplification double-stranded molecules to form single-stranded amplification molecules;  
 (d) recovering the single-stranded amplification molecules of step (c), and  
 (e) contacting the single-stranded amplification molecules with a fresh solid support;  
 (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules, and  
 (g) detecting the presence of the target molecules, wherein the detection of the single-stranded or double-stranded amplification molecules is indicative of the target molecules in a test sample.  
 
     
     
         32 . A method of detecting the presence or absence of one, or more, target nucleic acid molecules using a solid support comprising two or more immobilized oligonucleotide primers, of which at least one primer specifically hybridizes to the target nucleic acid molecules, comprising the steps of: 
 (a) hybridizing target nucleic acid molecules to a set of oligonucleotide primers immobilized on a solid support;    (b) thermocycling to amplify the target nucleic acid molecules hybridized to the oligonucleotide primers forming a double-stranded amplification molecule;    (c) cleaving and denaturing the double-stranded amplification molecule to form single-stranded amplification molecules;    (d) recovering the single-stranded amplification molecules of step (c),    (e) contacting the single-stranded amplification molecules with a fresh solid support, and    (f) repeating steps (a) through (e) to amplify the target nucleic acid molecules, and    (g) detecting the presence of the target molecules, wherein the detection of the single-stranded or double-stranded amplification molecules is indicative of the target molecules in a test sample.    
     
     
         33 . The method of  claim 32 , wherein the thermocycling step (b) comprises about 5 to about 50 thermocycles.  
     
     
         34 . The method of  claim 33 , wherein each thermocycle comprises about one minute each at 95° C., 60° C. and 72° C.  
     
     
         35 . The method of  claim 32 , wherein one or more single-stranded amplification molecules are labeled.  
     
     
         36 . The method of  claim 35 , wherein the label is selected from the group consisting of: radioactivity, chemiluminescence, luminescence and fluorescence.  
     
     
         37 . A kit for use in a solid-phase, multi-stage method of amplifying one, or more, target nucleic acid molecules comprising two or more stages of bridge amplification, wherein single-stranded amplification molecules produced in the first stage of bridge amplification initiates a second stage of bridge amplification, and each subsequent stage of bridge amplification is initiated with a single-stranded amplification molecule produced in the previous stage of bridge amplification, wherein one reagent comprises solid phase supports comprising at least one set of primers for amplifying at least one target nucleic acid molecule, said supports provided in a quantity sufficient for at least two stages of bridge amplification.  
     
     
         38 . The kit of  claim 37 , wherein the solid support reagent comprises beads wherein each bead comprises a set of primers specific for one or more target molecules.