Capture primers and capture sequence linked solid supports for molecular diagnostic tests

Abstract

The present invention provides systems, methods, and compositions for performing molecular tests. In particular, the present invention provides methods, compositions and systems for generating target sequence-linked solid supports (e.g., beads) using a solid support linked to a plurality of capture sequences and capture primers composed of a 3′ target-specific portion and a 5′ capture sequence portion. In certain embodiments, the target sequence linked solid support is used in sequencing methods (e.g., pyrosequencing, zero-mode waveguide type sequencing, nanopore sequencing, etc.) to determine the sequence of the target sequence (e.g., in order to detect the identity of a target nucleic acid in sample).

1 .- 30 . (canceled) 
     
     
         31 . A system comprising:
 a) at least one sequencing device; and   b) a primer pair comprising a capture primer and a reverse primer, wherein said capture primer comprises: i) a 3′ region configured to hybridize to a target nucleic acid such that it can be extended by a polymerase to form a first amplification product, and ii) a 5′ region comprising a capture sequence, wherein said 5′ region is configured to not hybridize to said target nucleic when said 3′ region of said capture primer is hybridized to said target nucleic acid, and wherein said reverse primer is configured to hybridize to said first extension product and be extended to form a second extension product.   
     
     
         32 . A system, comprising:
 a) a primer pair comprising a capture primer and a reverse primer, wherein said capture primer comprises: i) a 3′ region configured to hybridize to a target nucleic acid such that it can be extended by a polymerase to form a first amplification product, and ii) a 5′ region comprising a capture sequence, wherein said 5′ region is configured to not hybridize to said target nucleic when said 3′ region of said capture primer is hybridized to said target nucleic acid, and wherein said reverse primer is configured to hybridize to said first extension product and be extended to form a second extension product;   b) a reaction vessel or substrate; and   c) a detector configured to detect detectable signals produced in or on the reaction vessel or substrate, which detectable signals correspond to at least some nucleobases incorporated into a nucleic acid to generate nucleobase incorporation data.   
     
     
         33 . The system of  claim 32 , wherein the reaction vessel or substrate comprises at least one zero-mode waveguide. 
     
     
         34 . The system of  claim 32 , wherein the detector comprises a fluorescence microscope. 
     
     
         35 . A system, comprising:
 (a) a sequencing device configured to generate nucleic acid sequence data corresponding to the nucleic acid sequence of one or more amplicons produced using at least one purified oligonucleotide primer pair that comprises a capture primer and a reverse primer, wherein said capture primer comprises: i) a 3′ region configured to hybridize to a target nucleic acid such that it can be extended by a polymerase to form a first amplification product, and ii) a 5′ region comprising a capture sequence, wherein said 5′ region is configured to not hybridize to said target nucleic when said 3′ region of said capture primer is hybridized to said target nucleic acid, and wherein said reverse primer is configured to hybridize to said first extension product and be extended to form a second extension product; and   (b) a controller operably connected to said sequencing device, said controller configured to query a database with said nucleic acid sequence data to identify said target nucleic acid.   
     
     
         36 . A system, comprising:
 a) a capture primer, comprising:
 i) a 3′ region configured to hybridize to a target nucleic acid; and 
 ii) a 5′ region comprising a capture sequence; and 
   b) a first polymerase capable of extending said target nucleic acid to generate a first amplification product; and   c) a reverse primer configured to hybridize to said first amplification product;   d) a second polymerase capable of extending said first amplification product to generate a second amplification product, wherein said second amplification product comprises a 3′ capture sequence complement capable of hybridizing to said capture sequence;   c) a solid support comprising a plurality of bound capture sequences such that said 3′ capture sequence complement of said second amplification product hybridizes to at least one of said bound capture sequences to generate a hybridized solid support;   d) a third polymerase capable of extending at least one of said bound capture sequences along said second amplification product to generate a target sequence that is linked to said solid support;   e) a plurality of labeled oligonucleotides;   f) at least one primer nucleic acid that is at least partially complementary to at least a subsequence of said target sequence; and   g) at least one nucleotide incorporating biocatalyst capable of extending said primer nucleic acid to generate an extended primer nucleic acid that incorporates one or more of said labeled nucleotides at a terminal end of said extended primer nucleic acid.   
     
     
         37 . The system of  claim 36 , comprising a clonally amplified solid support comprising a plurality of said target sequences generated by a plurality of free capture sequences and a plurality of said reverse primers. 
     
     
         38 . The system of  claim 36 , further comprising a thermocycler capable of performing emulsion polymerase chain reaction (PCR) or bridge PCR. 
     
     
         39 . The system of  claim 36 , wherein said 5′ region is configured to not hybridize to said target nucleic when said 3′ region of said capture primer is hybridized to said target nucleic acid. 
     
     
         40 . The system of  claim 36 , further comprising at least one zero-mode waveguide. 
     
     
         41 . The system of  claim 36 , wherein said labeled oligonucleotides comprise different fluorescent labels. 
     
     
         42 . The system of  claim 36 , further comprising a fluorescence microscope. 
     
     
         43 . The system of  claim 36 , wherein at least one of said first polymerase, said second polymerase and said third polymerase is not the same polymerase. 
     
     
         44 . The system of  claim 36 , wherein said nucleotide incorporating biocatalyst comprises an enzyme selected from the group consisting of: a polymerase, a terminal transferase, a reverse transcriptase, a polynucleotide phosphorylase, and a telomerase. 
     
     
         45 . The system of  claim 36 , wherein said nucleotide incorporating biocatalyst comprises one or more modifications. 
     
     
         46 . The system of  claim 36 , wherein said label comprises a fluorescent dye, a non-fluorescent label, a colorimetric label, a chemiluminescent label, a bioluminescent label, a radioisotope, an antibody, an antigen, biotin, a hapten, or an enzyme. 
     
     
         47 . The system of  claim 36 , wherein said capture primer and said reverse primer are configured to hybridize with conserved regions of two or more different bioagents and flank variable regions of said two or more different bioagents. 
     
     
         48 . The system of  claim 36 , further comprising a database configured to compare a nucleic acid sequence of said target sequence to one or more database nucleic acid sequences to determine the organismal source of said target nucleic acid. 
     
     
         49 . The method of  claim 48 , wherein the organismal source is identified at one or more taxonomic rank levels selected from the group consisting of: a Domain, a Superphylum, a Superdivision, a Superclass, a Superorder, a Superfamily, a Superspecies, a Kingdom, a Phylum, a Division, a Class, a Legion, an Order, a Family, a Tribe, a Genus, a Species, a Subkingdom, a Subphylum, a Subclass, a Cohort, a Suborder, a Subfamily, a Subtribe, a Subgenus, a Subspecies, an Infrakingdom, a Branch, an Infraphylum, an Infraclass, an Infraorder, an Alliance, an Infraspecies, a Microphylum, a Pan/class, and a Parvorder. 
     
     
         50 . The system of  claim 36 , wherein said capture primer comprises a bar-code sequence between said 3′ region and said 5′ region. 
     
     
         51 . The system of  claim 36 , wherein a linker attaches a label to said labeled oligonucleotide.