US2005287591A1PendingUtilityA1

Nucleic acid amplification and detection method

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Assignee: GEN PROBE INCPriority: Jul 31, 2002Filed: Aug 24, 2005Published: Dec 29, 2005
Est. expiryJul 31, 2022(expired)· nominal 20-yr term from priority
Inventors:Kenneth A. Browne
B01J 2219/00313C40B 40/06B82Y 30/00B01J 19/0046C40B 50/14B01J 2219/00621B01J 2219/0061B01J 2219/00529C40B 30/04B01J 2219/00637B01J 2219/0063C07B 2200/11B01J 2219/00626B01J 2219/00608B01J 2219/00677B01J 2219/00576B01J 2219/00612C40B 60/14B01J 2219/005
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Claims

Abstract

Method of amplifying and detecting a nucleic acid target molecule contained in a test sample using a device having amplification primers and detectably labeled hybridization probes immobilized on the same surface of a solid support. Soluble amplicons produced in an in vitro nucleic acid amplification reaction employing the immobilized amplification primers are detected by the immobilized hybridization probes. Surface-immobilized amplification primers and hybridization probes are maintained in fluid communication throughout the procedure.

Claims

exact text as granted — not AI-modified
1 . A method of determining whether a test sample contains a target nucleic acid comprising a target sequence, said method comprising the steps of: 
 providing a device for amplifying nucleic acids and detecting amplification products, said device comprising, 
 a solid support having a surface,  
 a plurality of species of amplification primer immobilized to said surface, said plurality of species of amplification primer comprising a first amplification primer that comprises a sequence complementary to a first strand of said target nucleic acid, and  
 a plurality of species of detectably labeled hybridization probe immobilized to said surface, said plurality of species of detectably labeled hybridization probe comprising a first hybridization probe that comprises a detectable label and a sequence of bases complementary to said target sequence;  
   contacting said plurality of species of amplification primer and said plurality of species of detectably labeled hybridization probe with said test sample;    synthesizing in an in vitro amplification reaction multiple copies of said target sequence and the complement thereof by a process comprising enzymatic extension of said first amplification primer immobilized to said surface, whereby there are produced soluble amplification products if said test sample contained said target nucleic acid; and    detecting hybridization of said soluble amplification products to said first hybridization probe immobilized to said surface, thereby determining that said test sample contained said target nucleic acid, 
 wherein each of said plurality of species of detectably labeled hybridization probe and said plurality of species of amplification primer is in fluid communication with the others during the synthesizing and detecting steps.  
   
     
     
         2 . The method of  claim 1 , wherein no portion of said surface of said solid support is excluded from occupation by an immobilized oligonucleotide, said device having been manufactured by a process comprising immersion of said surface of said solid support in a liquid composition comprising immobilizable oligonucleotide primers.  
     
     
         3 . The method of  claim 2 , wherein said solid support is a bead.  
     
     
         4 . The method of  claim 2 , wherein said surface of said solid support is a planar surface.  
     
     
         5 . The method of  claim 4 , wherein said first hybridization probe is one component in an array.  
     
     
         6 . The method of  claim 2 , wherein the in vitro amplification reaction in the synthesizing step is an isothermal in vitro amplification reaction that does not require thermal cycling.  
     
     
         7 . The method of  claim 6 , wherein said process comprising enzymatic extension in the synthesizing step comprises enzymatic extension by a reverse transcriptase.  
     
     
         8 . The method of  claim 7 , wherein said soluble amplification products consist of RNA.  
     
     
         9 . The method of  claim 7 , wherein said reverse transcriptase comprises an endogenous RNase H activity.  
     
     
         10 . The method of  claim 2 , wherein said plurality of species of detectably labeled hybridization probe comprises no more than two species of detectably labeled hybridization probe.  
     
     
         11 . The method of  claim 10 , wherein said plurality of species of detectably labeled hybridization probe that comprises no more than two species of detectably labeled hybridization probe comprises no more than a single species of detectably labeled hybridization probe.  
     
     
         12 . The method of  claim 2 , wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′ terminus available for extension by a DNA polymerase activity.  
     
     
         13 . The method of  claim 12 , wherein said plurality of species of detectably labeled hybridization probe comprises no more than two species of detectably labeled hybridization probe.  
     
     
         14 . The method of  claim 11 , wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′ terminus available for extension by a DNA polymerase activity.  
     
     
         15 . The method of  claim 2 , wherein each of said plurality of species of detectably labeled hybridization probe in said device comprises a fluorophore moiety and a quencher moiety.  
     
     
         16 . The method of  claim 15 , wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular beacon.  
     
     
         17 . The method of  claim 15 , wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular torch.  
     
     
         18 . The method of  claim 1 , wherein said plurality of species of detectably labeled hybridization probe comprises no more than two species of detectably labeled hybridization probe.  
     
     
         19 . The method of  claim 18 , wherein said plurality of species of detectably labeled hybridization probe that comprises no more than two species of detectably labeled hybridization probe comprises no more than a single species of detectably labeled hybridization probe.  
     
     
         20 . The method of  claim 19 , wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′ terminus available for extension by a DNA polymerase activity.  
     
     
         21 . The method of  claim 18 , wherein said plurality of species of amplification primer comprises no more than a single species of amplification primer having a free 3′ terminus available for extension by a DNA polymerase activity.  
     
     
         22 . The method of  claim 18 , wherein said solid support is a bead.  
     
     
         23 . The method of  claim 18 , wherein said surface of said solid support is a planar surface.  
     
     
         24 . The method of  claim 1 , wherein said process comprising enzymatic extension in the synthesizing step comprises enzymatic extension by a reverse transcriptase.  
     
     
         25 . The method of  claim 1 , wherein the in vitro amplification reaction in the synthesizing step is an isothermal in vitro amplification reaction that does not require thermal cycling.  
     
     
         26 . The method of  claim 25 , wherein said process comprising enzymatic extension in the synthesizing step comprises enzymatic extension by a reverse transcriptase.  
     
     
         27 . The method of  claim 26 , wherein said soluble amplification products produced in the synthesizing step consist of RNA.  
     
     
         28 . The method of  claim 26 , wherein said reverse transcriptase comprises an endogenous RNase H activity.  
     
     
         29 . The method of  claim 1 , wherein each of said plurality of species of detectably labeled hybridization probe in said device comprises a fluorophore moiety and a quencher moiety.  
     
     
         30 . The method of  claim 29 , wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular beacon.  
     
     
         31 . The method of  claim 29 , wherein each of said plurality of species of detectably labeled hybridization probe that comprises a fluorophore moiety and a quencher moiety is a molecular torch.  
     
     
         32 . The method of  claim 1 , wherein the detecting step comprises detecting a fluorescent signal.  
     
     
         33 . The method of  claim 1 , wherein said soluble amplification products produced in the synthesizing step consist of RNA.  
     
     
         34 . The method of  claim 1 , wherein said solid support is a bead.  
     
     
         35 . The method of  claim 1 , wherein said surface of said solid support is a planar surface.  
     
     
         36 . The method of  claim 35 , wherein said first hybridization probe is one component in an array.

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