Abstract:
This invention combines artificially expanded genetic information systems (AEGIS) with self-avoiding molecular recognition systems (SAMRS), in processes that involve template-directed primer extension in highly multiplexed form in mixtures containing large numbers of primers. This process yields extension products, or in its PCR format, amplicons, that have AEGIS tags that can be cleanly captured in highly complex mixtures.

Description:
Continuation in part of U.S. patent application Ser. No. 12/229,159 filed 2008 Aug. 20 Which is a Continuation in part of U.S. patent application Ser. No. 11/647,609 filed 2008 Dec. 30 Which is a Continuation in part of U.S. patent application Ser. No. 11/271,366 filed 2005 Nov. 12 Which is based on provisional patent applications 60/627,460 and 60/627,459 filed 2004 Nov. 13 and 60/654,424 filed 2005 Dec. 20 
    
    
     This invention was made with government support under W911NF12C0059 awarded by The Office of the Secretary of Defense US Army RDECOM ACQ CTR. The government may have certain rights in the invention. 
    
    
     FIELD 
     This invention relates to the field of nucleic acid chemistry, more specifically to the field of compositions and processes that can serve as primers for the copying of DNA and RNA. Most specifically, this invention relates to compositions of matter that bind to natural DNA and RNA following simple rules as they serve as primers, without binding as strongly to themselves. 
     BACKGROUND 
     Scientists have long sought innovative molecular recognition systems that have binding properties that are useful in different ways. The structures of these systems have been modeled to resemble the structures of DNA and RNA which, in their polymeric form, are called “oligonucleotides”. Further, as with DNA and RNA, the molecular recognition systems have been useful because they bind to other components of the molecular recognition systems and/or to natural DNA and RNA following rules that can be expressed in a form that guides practitioners of ordinary skill in the art and enables them to do useful things. 
     DNA serves as an archetype to illustrate both molecular structure and rule base recognition. With DNA, three rules (A pairs with T, G pairs with C, the strands are antiparallel) permit the design of two DNA molecules that bind to each other in aqueous solution. When the rules are perfectly followed, two perfectly complementary DNA strands of a substantial length (15-20 nucleotides is normally sufficient in physiological buffers at 37° C.) will bind to each other with substantial selectivity even in complex mixtures containing many other DNA molecules. Heuristic rules have been developed over the years to permit the prediction of general trends in DNA:DNA binding affinity. These have come by performing substantial numbers of melting temperature experiments. For examples as heuristic rules, longer DNA strands generally bind to their partners with higher melting temperatures (T m s) than shorter strands. G:C pairs generally contribute more to duplex stability than A:T pairs. More highly parameterized models improve on the estimates of melting temperatures [All98a] [All98b] [Mar85] [Mat98]. While it remains true that the precise stability of duplexes may not be predictable, that imprecision does not defeat the utility of DNA:DNA binding or require undue experimentation to exploit, even though the number of different DNA sequences of length n (=4 n ) that would fall within a patent for the DNA molecular recognition system would be enormous. 
     It has been argued that this rule-based behavior arises because of the repeating charge in the backbone of nucleic acids [Ben04]. Certainly, analogs that have that repeating charges in their backbone maintain their rule-based pairing behavior even if they become quite long. In contrast, the few examples of useful nucleic acid analogs that lack a repeating charge in their backbone do not maintain their rule-based binding behavior in polymers built from two-dozen or more monomer units (fewer if the nucleobases are predominately guanine). The archetypal example of such an uncharged DNA analog is the peptide nucleic acids (PNAs) [Egh92], where rule-based molecular recognition does not survive in longer molecules. 
     Artificially Expanded Genetic Information Systems (AEGIS) 
     An archetype of a human-invented rule-based molecular recognition is the artificially expanded genetic information system (AEGIS) disclosed in U.S. Pat. No. 5,432,272. The design of this artificial molecular recognition system began with the observation that two principles of complementarity govern the Watson-Crick pairing of nucleic acids: size complementarity (large purines pair with small pyrimidines) and hydrogen bonding complementarity (hydrogen bond donors from one nucleobase pair with hydrogen bond acceptors from the other). These two principles give rise to the simple rules for base pairing (“A pairs with T, G pairs with C”) that underlie genetics, molecular biology, and biotechnology. 
     U.S. Pat. No. 5,432,272 pointed out that these principles can be met by nucleotides other than adenine (A) and thymine (T), and guanine (G) and cytosine (C). Rather, twelve nucleobases forming six base pairs joined by mutually exclusive hydrogen bonding patterns might be possible within the geometry of the Watson-Crick base pair.  FIG. 1  shows some of the standard and non-standard nucleobase pairs, together with the nomenclature to designate them. Those nucleobase analogs presenting non-standard hydrogen bonding patterns are part of an Artificially Expanded Genetic Information System, or AEGIS. 
     U.S. Pat. No. 5,432,272 and subsequent patents all taught that the hydrogen bonding pattern that makes an AEGIS component useful as a unit of molecular recognition is distinguishable from the heterocycle that implements it. This means that different heterocycles can often serve interchangeably as molecular recognition elements. This, in turn, permits the elements of an artificial molecular recognition system to be chosen based on considerations other than simple recognition. Thus, the pyADA hydrogen bonding pattern in AEGIS is implemented by thymidine, uridine, uridine derivatives carrying a 5-position linker attached to a fluorescent moiety, uridine derivatives carrying a 5-position linker attached to a biotin, and pseudouridine, for example. 
     Four features of the AEGIS system make it suited for application:
     (a) AEGIS supports rule-based design. Anyone of ordinary skill in the art can design two AEGIS-containing molecules that bind to each other, after learning only a few additional rules, just as they can design binding partners with standard DNA. Again, a critical mass of melting temperatures were collected to support heuristic rules that allow prediction of affinity. As with DNA, the precise T m s are not predictable even with these heuristic rules, but this imprecision does not defeat the utility of the system, or create a need for undue experimentation to design AEGIS pairing partners.   (b) This rule-based molecular recognition displayed by AEGIS is orthogonal to that displayed by standard DNA. If two strands incorporating standard DNA bases are mixed with two other strands incorporating AEGIS components, the first pair will bind to each other only, and the second pair will bind to each other only, without formation of hybrids between the strands containing canonical and non-canonical bases. This allows two molecular recognition processes to occur independently in the same vessel.   (c) Sequences built from AEGIS components have higher information density (more different sequences per unit length), especially when they incorporate the full 12 letters that the AEGIS technology allows. This allows fewer near-mismatches in complicated systems to slow hybridization, for example. Thus, AEGIS tags hybridize more quickly [Col97].   (d) Enzymes can be found that allow AEGIS systems to be manipulated in ways common in biotechnology with standard DNA. These enzymes include polymerases that do primer extension, copy templates that contain AEGIS components, and amplify AEGIS oligonucleotides a polymerase chain reaction (PCR). Here, undue experimentation is often required to obtain enzymes that do this effectively, as many natural enzymes regard non-standard nucleotides as “foreign”, and do not accept them or, if they do, do not accept them with useful affinity.   

     An archetypal application of AEGIS is in the branched DNA (bDNA) assay used to measure levels of HIV, hepatitis B, and hepatitis C viruses in human patients [Elb04a][Elb04b)]. As this example shows, even though the behavior of DNA duplexes built from AEGIS components having different sequences are not identical and may not be precisely predictable, this has not prevented the AEGIS molecular recognition system from improving the health care of some 400,000 patients annually [Ben04]. This is an illustration of the utility of orthogonality in the analytical chemistry of nucleic acids. 
     Self Avoiding Molecular Recognition Systems (SAMRS) 
     A self-avoiding molecular recognition system (SAMRS) has components that bind to natural DNA or RNA, but not to other components of the same unnatural system. In its general description, a SAMRS incorporates nucleobase analogs that replace T, A, G, and C by analogs that are indicated as T*, A*, G*, and C*, which are collectively called “* analogs” of T, A, G, and C respectively. In the simplest implementation of this concept, these * analogs are each able to form two hydrogen bonds to the complementary A, T, C, and G. This means that the T*:A, A*:T, C:*G, and G*:C nucleobase pairs contribute to duplex stability to approximately the same extent as an A:T pair. A SAMRS obtains its self-avoiding properties because the hydrogen bonding groups of the * analogs are chosen the T*:A* and C*:G* nucleobase pairs do not contribute as much to duplex stability because (in the simplest implementation) they are joined by only one hydrogen bond. 
     As with standard DNA, standard RNA, and oligonucleotides that add non-standard nucleobase pairing, within predicting the binding properties of any sequence within a SAMRS system will be subject to the same imprecision as predicting the properties of an arbitrary DNA or RNA molecule. Thus, as a general rule, if individuals of ordinary skill in the art wish to design a SAMRS sequence that binds to a preselected standard DNA molecule with a Tm of 25° C., they would write down the preselected sequence in the 5′-to-3′ direction, and then write below the SAMRS sequence in an antiparallel direction, matching a T* against every A in the preselected sequence, an A* against every T in the preselected sequence, a C* against every G in the preselected sequence, and a G* against every C in the preselected sequence. It is an open question as to whether such simple instructions allow one of ordinary skill in the art to obtain useful outcomes without undue experimentation. As elaborated below, attempts to obtain such utility failed when we took instruction from the prior art. One object of the instant invention is to provide SAMRS components that provide utility based on precisely this simple a set of rules and instructions. 
     The need for self-avoiding behaviors has long been pressing when an experimentalist sought to have mixtures containing more than two oligonucleotides, and especially pressing when making libraries of oligonucleotides (defined as having 10 or more oligonucleotide components), especially when those oligonucleotides were to interact with enzymes such as DNA polymerases. This problem is exemplified by multiplexed PCR, where the amplification is sought of many segments of DNA in one pot. This is attempted by adding in large excess two primers flanking each segment, contacting mixture with nucleoside triphosphates, and cycling the mixture up and down in temperature in the presence of a thermostable DNA polymerase. At low temperatures, the primers anneal to the template. At higher temperatures, the polymerase extends the primer to make a product copy of the template. At the highest temperature, the product copy falls off the template, allowing more primers to bind when the temperature is dropped. The primers compete with full length product copies for their binding sites on the template by being present in high concentrations. 
     While PCR can be successfully multiplexed up to a dozen or so amplicons, with careful design to avoid having the primers present in high concentrations interact with each other, eventually even the most careful design does not prevent primer-primer interactions. These create undesired amplicons, primer dimers, and other artifacts that defeat the utility of the PCR. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . An “artificially expanded genetic information system (AEGIS). Twelve nucleobases in a nucleic acid alphabet that form specific pairs with the constraints of the Watson-Crick geometry. Pyrimidine base analogs are designated “py”, purine by “pu”. Upper case letters following a designation indicate the hydrogen bonding pattern of acceptor (A) and donor (D) groups. Thus, cytosine is pyDAA. 
         FIG. 2 . Another “artificially expanded genetic information system (AEGIS). Twelve nucleobases in a nucleic acid alphabet that form specific pairs with the constraints of the Watson-Crick geometry. Note different implementations of the same hydrogen bonding pattern in many cases, including the addition of an amino group to complete the hydrogen bonding pattern of standard adenosine, by making the nucleobase diaminopurine. 
         FIG. 3 . Self-Avoiding Molecular Recognition System (SAMRS) in their presently preferred implementation. A molecular recognition system that binds to complementary natural DNA, but not to complementary SAMRS sequences. The pairing of each of the complements of the SAMRS heterocycles (denoted by an asterisk *) with a standard nucleobase is joined by two hydrogen bonds, while pairs between any two size-complementary SAMRS components are joined by (at most) one hydrogen bond. Note that the G*-C* and A*-T* pairs in the wobble structure do not have two productive hydrogen bonds. Diaminopurine (not shown) is a presently preferred alternative implementation of A*. 
     
    
    
     SUMMARY OF THE INVENTION 
     This invention combines AEGIS with SAMRS, where a tag containing one or more AEGIS nucleotides is appended to the 5′-end of the molecules that are claimed in the parent application, U.S. patent application Ser. No. 12/229,159, which is incorporated herein in its entirety by reference. This tag has utility, for example, by allowing multiplexed primer extension that is directed by templates built from only natural nucleotides to yield products that have tags that contain one or more AEGIS nucleotides, and therefore do not fully complement any natural oligonucleotide. It also has utility in a process for multiplexed nested PCR of multiple targets [Bro97], where the product amplicons carry one or more tags that contain one or more AEGIS nucleotides, and therefore do not fully complement any natural oligonucleotide. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The goal of the instant invention is to provide primers that could be extended by DNA polymerases when templated on a natural DNA, and to provide primers that could support PCR (which requires that a primer, after being extended, must also be accepted as a template by a DNA polymerase), where the products of these processes have an AEGIS tag. 
     With both SAMRS and AEGIS, the instant invention teaches a distinction between the hydrogen bonding pattern of a SAMRS system and the heterocycle used to implement it. As is well known in the art, appendages may be attached the 5-position of pyrimidines without interfering with the hydrogen bonding that supports duplex formation. Indeed, 5-position alkyl, allyl, and acetylenic substituents at those positions generally encourage duplex formation. Likewise, substituents at this position may carry tags useful for capture (such as biotin) or detection (such as fluorescent species). The instant invention teaches that similar substituents can be attached at the “5-equivalent” position of the heterocycle that implements the SAMRS and AEGIS, noting that the IUPAC numbering of the heterocycle may assign a different numbering to the 5-equivalent position of any given heterocycle. 
     Analogous substitutions may be placed at the 7-equivalent position of a 7-deazapurine analog that is a part of a SAMRS and AEGIS. Further, the 7-equivalent nitrogen may be replaced by a CH unit simply to prevent Hoogsteen binding. 
     Likewise, while 2′-deoxyribose is the preferred backbone when it is desired to have the SAMRS component be recognized by natural DNA polymerases, RNA polymerases, and reverse transcriptases, tighter binding is obtained by placing the SAMRS- and AEGIS-enabling heterocycles on 2′-OMe, 2′-O-alkyl, and/or 2′-O-allyl ribose, PNA, or LNA, which are all taught here as part of the instant invention (such disclosure not being obvious without such a teaching). 
     The discussion of the inventive steps by which the presently preferred implementations of the SAMRS concept were developed is provided in U.S. patent application Ser. No. 12/229,159, of which this is a continuation-in-part. U.S. patent application Ser. No. 12/229,159 is incorporated in its entirety by reference. The presently preferred implementations of the SAMRS heterocycles are in  FIG. 3 . 
     As noted in U.S. patent application Ser. No. 12/229,159, reduction to practice discovered as an unexpected phenomenon that the melting temperatures of duplexes supported by only base pairs joined by two hydrogen bonds were abnormally low. Thus, while the 2-thioT:A pair was modestly more stable on average (with the metric being a higher Tm in a variety of contexts) than the T:A pair, a fact well known in the literature, and the I:C pair was significantly less stable than the G:C pair (a fact also well known), duplexes joined by only 2-thioT:A, 2-AP:T, I:C, and  4Et C:G pairs were significantly less stable than expected. 
     This observation prompted the exploration of primers having the self-avoiding property at the 3′-end of the primer more than at the 5′-end of the primer, as it is overlap of the 3′-ends of primers in primer libraries that causes primer-primer interactions that defeat the PCR analysis. Thus, this would direct one of ordinary skill in the art to place standard nucleobases at the 5′-end. 
     As a consequence, rules were developed that give the presently preferred embodiments for the primer segments. The preferred 5′-end of the primer is a moiety commonly used in primers, including without limitation OH (the 5′-OH group is free), O-phosphate (allowing the 5′-end to be ligatable), O-oligonucleotide, —NH 2 , or a phosphate or an amino group linked to a biotin or a fluorescent tag. The 3′-terminal nucleotide preferably has one of the standard nucleotides, adenine (or diaminopurine), thymine, guanine, cytosine, or uracil, or one of the A*, T*, G*, and C* nucleobases, with the most preferred application being a standard nucleotide at the 3′-end. The SAMRS-containing segment next in from the 3′-end is preferably 4 to 6 nucleotides in length, and entirely composed of A*, T*, G*, and C* nucleotides, although a single standard nucleotide can be in segment. The next segments, proceeding away from the 3′-end, are presently preferred to be constructed exclusively from A, T, G, and C, although single SAMRS nucleotides in this region can function as well, preferably if they are thiothymidine or thiouracil. This segment is chosen to give a desired affinity to its complement, and is preferably 10 to 20 nucleotides long. In any case, the presently preferred sum of these two segments primers is at least 15, so as to achieve useful affinity to a target oligonucleotide. 
     These segments, including the 3′-nucleotide, the SAMRS-rich segment, and the SAMRS-poor segments, are designed to be substantially complementary to a portion of the sequence of a target oligonucleotides, to which it will hybridize in the claimed process. 
     The preferred AEGIS tag contains nucleobases independently selected from the group consisting of A, T, G, C, K, X, V, J, S, B, Z and P, wherein K, X, V, J, S, B, Z and P are the nucleobases disclosed in  FIG. 1  or  FIG. 2 . The tag must contain at least one K, X, V, J, S, B, Z and P, but more preferably it contains at least two, and is preferably 5 to 30 nucleotides long. 
     EXAMPLES 
     Example 1 
     Multiplexed Detection of Mosquito-Borne Arboviruses 
     Mosquito-borne arboviruses must be detected in public health surveillance environments. This example combined the self avoiding molecular recognition system (SAMRS), which enables high levels of multiplexing, with an artificially expanded genetic information system (AEGIS), which enables very clean PCR amplification in nested PCR formats. Luminex “liquid microarrays” were exploited for downstream multiplexed detection. 
     Targets 
     This example showed this combination supporting single-tube PCR amplification assays to seek RNA from 21 mosquito-borne RNA viruses from the genera Flavivirus, Alphavirus, and Orthobunyavirus. This assay differentiated between many closely-related viral targets, including dengue, West Nile, Japanese encephalitis, and the California serological group viruses. 
                                   TABLE 1                   Viruses targeted.            Family/Genus   Viruses and abbreviations   Primer identity               Flaviviridae/   West Nile (WN)   Forward-WNm1, Reverse-WNm1         Flavivirus     Japanese encephalitis (JE)   Forward-JE m1, Reverse-JE m1       Group IV,   Saint Louis encephalitis (SLE)   Forward-SLEVm1, Reverse-SLEVm1       positive ssRNA   Yellow fever (YF)   Forward-YF m3, Reverse-YF m3           Dengue serotype 1 (D1)   Forward-D1, Reverse-Den (1, 3)           Dengue serotype 2 (D2)   Forward-D2, Reverse-D (2, 4)           Dengue serotype 3 (D3)   Forward-D3, Reverse-D (1, 3)           Dengue serotype 4 (D4)   Forward-D4, Reverse-D (2, 4)           Murray valley encephalitis (MVE)   Forward-MVE, Reverse-MVE           Rocio (Rocio)   Forward-Rocio, Reverse-Rocio       Togaviridae/   Eastern Equine Encephalitis (EEE)   Forward-EEEm1, Reverse-EEEm1         Alphavirus     Venezuelan Equine Encephalitis (VEE)   Forward-VEEm1, Reverse-VEEm1       Group IV,   Western Equine Encephalitis (WEE)   Forward-WEEm1, Reverse-WEEm1       positive ssRNA               Bunyaviridae/   California encephalitis (CE)   Forward-CE, Reverse-CE         Orthobunyavirus     Jamestown Canyon   Forward-JTC Reverse-JTC       Group V   La Crosse encephalitis (LAC)   Forward-LAC, Reverse-LAC       negative ssRNA   Keystone (KS)   Forward-KS, Reverse-KS           Snowshoe Hare (SSH)   Forward-SSH, Reverse-SSH           San Angelo (SA)   Forward2-CAcom, Reverse 1-CAcom           Serra do Navio (SN)   Forward2-CAcom, Reverse 1-CA-com           Melao (Mel)   Forward-Mel, Reverse-Mel                    
Primers and Probes
 
     Primers and capture probes containing artificial SAMRS and AEGIS nucleotides (Table 2) were synthesized on ABI 394 and ABI 3900 synthesizers in-house. Primers and capture probes were designed to complement a majority of the strains from each of the target viruses. For the simulants, ssDNA oligonucleotides (Amplimers, Appendix A. Supplementary data Table 2) were chosen arbitrarily to represent a single strain. 
                                       TABLE 2                   Hybrid SAMRES-AEGIS primers and AEGIS (APTC) probes used in this study.       All reverse primers are 5′-biotinylated; the probes are 5′-amino-       C12- modified. The AEGIS tags in the primers are underlined.            Oligos       Genome   GB       primers/probes   Sequences 5′-3′   Region   Accession No.               Forward WNm1     CTAPTCCPCCAPCPAPC     163-181   NC-009942       primer   CGCGTGTTGTCCTTG*A*T*T*G       SEQ ID NO 1               Reverse WNm1     CAGPAAGPGGTPGPTPG     312-293   NC-009942       primer   CACACCTCTCCATCGA*T*C*C*A       SEQ ID NO 2               WN probe   APPTTCACAPCAATTPCTCC   259-278   NC-009942                   SEQ ID NO 3               Forward JE m1     CTAPTCCPCCAPCPAPC     10612-10628   NC_001437       primer   GACCAACGTCAGG*C*C*A*C       SEQ ID NO 4               Reverse JE m1     CAGPAAGPGGTPGPTPG     10769-10748   NC_001437       primer   GGGTCTCCTCTAACCTCT*A*G*T*C       SEQ ID NO 5               JE probe   CACPPCCCAAPCCTCPTCTA   10705-10724   NC_001437                   SEQ ID NO 6               Forward SLE     CTAPTCCPCCAPCPAPC     10561-10577   NC_007580       m1 primer   TGGCACGTAGGCT*G*G*A*G       SEQ ID NO 7               Reverse SLEm1     CAGPAAGPGGTPGPTPG     10634-10614   NC_007580       primer   CAGACAGCACCTTTAGC*A*T*G*C       SEQ ID NO 8               SLE probe   CAPACCAPAAATPCCACCT   10591-10610   NC_007580                   SEQ ID NO 9               Forward YF m3     CTAPTCCPCCAPCPAPC     25-44   NC_002031       primer   GTGCATTGGTCTGCAA*A*T*C*G       SEQ ID NO 10               Reverse YF m3     CAGPAAGPGGTPGPTPG     164-146   NC_002031       primer   CCATATTGACGCCCA*G*G*G*T       SEQ ID NO 11               YF probe   PAPCPATTAPCAPAPAACTPAC   91-112   NC_002031                   SEQ ID NO 12               Forward D1     CTAPTCCPCCAPCPAPC     105-127   FJ639679.1       primer   GTCTTTCAATATGCTGAAA*C*G*C*G       SEQ ID NO 13               Forward D2     CTAPTCCPCCAPCPAPC     10433-10452   EU482570.1       primer   GAGGCCACAAACCATG*G*A*A*G       SEQ ID NO 14               Forward D3     CTAPTCCPCCAPCPAPC     103-128   EU482596.1       primer   GTCTATCAATATGCTGAAA*C*G*C*G       SEQ ID NO 15               Forward D4     CTAPTCCPCCAPCPAPC     10363-10379   GQ199883.1       primer   ATGCGCCACGGAA*G*C*T*G       SEQ ID NO 16               Reverse D (1,3)     CAGPAAGPGGTPGPTPG     174-152 (D1)   FJ639679.1       primer   TGAGAATCTCTTCGCCAAC*T*G*T*G       SEQ ID NO 17               Reverse D (2,4)     CAGPAAGPGGTPGPTPG     10497-10479   EU482570.1       primer   GGAGGGGTCTCCTCT*A*A*C*C   (D2)   SEQ ID NO 18               D1 probe   CPAPAAACCPCPTPTCAACT   128-147   FJ639679.1                   SEQ ID NO 19               D2 probe   CPCATPPCPTAPTPPACTAP   10457-10476   EU482570.1                   SEQ ID NO 20               D3 probe   APAAACCPTPTPTCAACTPP   131-151   EU482596.1                   SEQ ID NO 21               D4 probe   PCPTPPCATATTPPACTAPC   10383-10402   GQ199883.1                   SEQ ID NO 22               Forward MVE     CTAPTCCPCCAPCPAPC     535-551   NC_000943       primer   TGATCGCCATTCC*A*A*C*C       SEQ ID NO 23               Reverse MVE     CAGPAAGPGGTPGPTPG     614-594   NC_000943       primer   GGTGTCATCACACATAA*A*T*C*C       SEQ ID NO 24               MVE probe   PTCPPATTCPAPCCATTPAC   571-590   NC_000943                   SEQ ID NO 25               Forward-Rocio     CTAPTCCPCCAPCPAPC     1883-1903   AY632542       primer   CAAGAACCCAGTTGACA*C*A*G*G       SEQ ID NO 26               Reverse-Rocio     CAGPAAGPGGTPGPTPG     2036-2015   AY632542       primer   GGGAACAAATGGATTGAC*C*G*T*C       SEQ ID NO 27               Rocio probe   PAPAACCTACATPATCTCACTCC   1977-1999   AY632542                   SEQ ID NO 28               Forward-     CTAPTCCPCCAPCPAPC     11034-11057   NC_003899       EEEm1 primer   CTGAGAGCGGATCATTTACA*T*T*C*C       SEQ ID NO 29               Reverse-     CAGPAAGPGGTPGPTPG     11133-11111   NC_003899       EEEm1 primer   CAATCTCCTTTGCAGGTAA*C*T*G*C       SEQ ID NO 30               EEE probe   PCTTTTAAPCTPCAPPTCTPC   11084-11104   NC_003899                   SEQ ID NO 31               Forward-     CTAPTCCPCCAPCPAPC     4339-4360   NC_001449       VEEm1 primer   CAGTAGCGATTCCACTGT*T*G*T*C       SEQ ID NO 32               Reverse-     CAGPAAGPGGTPGPTPG     4485-4462   NC_001449       VEEm1 primer   GAGTCATTTCCCATTTCTTG*T*C*C*C       SEQ ID NO 33               VEE probe   PCTPACAPCTTTAPACACCAC   4415-4435   NC_001449                   SEQ ID NO 34               Forward-     CTAPTCCPCCAPCPAPC     345-366   NC_003908       WEEm1 primer   CAAGAACATAGCCTCTAA*G*G*C*G       SEQ ID NO 35               Reverse-     CAGPAAGPGGTPGPTPG     482-460   NC_003908       WEEm1 primer   GCGTACACATCTTGGTATA*C*T*G*C       SEQ ID NO 36               WEE probe   TPTATPCACACAPACPCCAC   418-437   NC_003908                   SEQ ID NO 37               Forward-CE     CTAPTCCPCCAPCPAPC     675-694   U12800       primer   CGGCATGATTGCAAAG*A*G*T*C       SEQ ID NO 38               Reverse-CE     CAGPAAGPGGTPGPTPG     792-770   U12800       primer   CGGAGCTTATGGCAACTTT*A*T*C*C       SEQ ID NO 39               CE probe   PTTTPAPCPACACTPCTAPAAC   731-752   U12800                   SEQ ID NO 40               Forward JTC     CTAPTCCPCCAPCPAPC     283-304   EF681804       primer   CAACGATCTTACCATCCA*T*C*G*G       SEQ ID NO 41               Reverse JTC     CAGPAAGPGGTPGPTPG     435-412   EF681804       primer   CCATTGTTCCAATGAATGCC*A*T*T*G       SEQ ID NO 42               JTC probe1   CAPAPAPAACTCATAAPPAPCAC   365-387   EF681804                   SEQ ID NO 43               JTC probe2   PCACCATCATAAATCCAATTPCAPA   384-408   EF681804                   SEQ ID NO 44               Forward LAC     CTAPTCCPCCAPCPAPC     577-597   NC_004110       primer   CACAGAGTCAAGCAAGG*C*A*T*G       SEQ ID NO 45               Reverse LAC     CAGPAAGPGGTPGPTPG     736-715   NC_004110       primer   GGCCTCCTTTTCCCCATT*T*A*A*G       SEQ ID NO 46               LAC probe   PATPTCACAPAAPPTTPCAPC   663-683   NC_004110                   SEQ ID NO 47               Forward KS     CTAPTCCPCCAPCPAPC     376-396   U12801       primer   GTGAGGACGAGTCACAA*A*A*G*G       SEQ ID NO 48               Reverse KS     CAGPAAGPGGTPGPTPG     476-453   U12801       primer   GAGATAGATTTCTACACCGT*T*G*C*C       SEQ ID NO 49               KS probe   PATCAAPAPCACTPTCATCAATCC   401-424   U12801                   SEQ ID NO 50               Forward SSH     CTAPTCCPCCAPCPAPC     687-707   J02390       primer   CCAAGAGCCTGAAGGAA*G*T*A*G       SEQ ID NO 51               Reverse SSH     CAGPAAGPGGTPGPTPG     793-772   J02390       primer   CCTTACTTATGGGAGCCT*G*A*T*G       SEQ ID NO 52               SSH probe   PACACTPCCAPATCATTCTTPC   740-761   J02390                   SEQ ID NO 53               Forward 2 CA     CTAPTCCPCCAPCPAPC     112-132 (SA)   U47139       common primer   CGGTGCAAATGGATTTG*A*T*C*C       SEQ ID NO 54               Forward 2 CA     CTAPTCCPCCAPCPAPC     111-131 (SN)   U47140       common primer   CGGTGCAAATGGATTTG*A*T*C*C       SEQ ID NO 54               Reverse 1 CA     CAGPAAGPGGTPGPTPG     235-216 (SA)   U47139       common primer   GAGAGCAGCTTTGGCT*T*T*T*G       SEQ ID NO 55               Reverse 1 CA     CAGPAAGPGGTPGPTPG     234-215 (SN)   U47140       common primer   GAGAGCAGCTTTGGCT*T*T*T*G       SEQ ID NO 55               SA probe   CPATCAPTTTPTCTTCAPTTAPPATC   174-199   U47139                   SEQ ID NO 56               SN probe   CTTACAPCCPTTAPAATCTTCTTCC   181-205   U47140                   SEQ ID NO 57               Forward Mel     CTAPTCCPCCAPCPAPC     659-679   U12802       primer   CTGAAGGATGTAGAGCA*G*C*T*G       SEQ ID NO 58               Reverse Mel     CAGPAAGPGGTPGPTPG     777-755   U12802       primer   GCCGAATTCATTAGAGGAC*C*A*T*C       SEQ ID NO 59               Mel probe   capaapttcpptpttapacttcc   725-747   U12802                   SEQ ID NO 60                    
PCR and Simulant Preparation
 
     PCR targeting RNA virus simulants was set up in 1× JumpStart reaction buffer (10 mM Tris-HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl 2 ; 0.001% (w/v) gelatin) (Sigma-Aldrich, St. Louis, Mo.). The other components of the reaction mixture were (in a total volume of 100 μL): 2.5 ng/μL DNA oligo; 0.4 mM dNTPs; 0.4 μM each, Forward T7 primer and Reverse target-specific primer; JumpStart Taq DNA polymerase (2 units, Sigma), nuclease-free ddH 2 O (added to create a final volume of 100 μL). After the initial denaturation at 95° C. for 2 minutes, 35 cycles of amplification were performed (94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1 minute). A final extension cycle was run at 72° C. for 5 minutes. Each PCR product (in 100 μL) was ethanol-precipitated and dissolved in nuclease-free dd H 2 O (12 μL). The resulting PCR products were sequenced in both directions (University of Florida, ICBR) and 5 pmol (about 2 μL) of the concentrated PCR product was used as a T7-DNA template to make RNA simulants. 
     For simulant production, a T7 RNA polymerase-dependent transcription reaction mixture (20 μL) was set up in a 1× transcription buffer (40 mM Tris, pH7.8, 20 mM NaCl, 18 mM MgCl 2 , 2 mM spermidine HCl, 10 mM DTT). The reaction mixture contained ATP, CTP, GTP, and UTP (75 mM stock concentration 2 μL each), DNA template (2.5-5 pmol, purified and concentrated PCR product); T7 RNA polymerase (2 μL of a 200 U/μL to give 20 U/μL final concentration). Reaction mixtures were incubated at 37° C. for 8-12 hours. Turbo DNase was then added (2 U per reaction mixture, Life Technologies) to remove DNA template. The mixtures were then incubated at 37° C. for 15-20 minutes. RNA products were isolated by phenol-chloroform extraction and dissolved in nuclease-free water (20 μL). RNA products were resolved by 3% TBE agarose-gel electrophoresis and quantitated by their UV absorbance at 260 nm. The purity of RNAs was evaluated from their A260/A280 ratio. For pure RNA, a ratio of 1.8-2.1 is expected. The absence of template DNA in the RNA samples was confirmed by conventional PCR with Platinum Taq DNA polymerase (Life Technologies) and the ethidium-bromide gel. Samples were aliquoted and kept at −80° C. 
     Monoplex PCRs were first performed using each target RNA simulant separately to assess the efficacy of the primers in PCR cycling, as well as to determine the sensitivity of the assay. Reactions were then optimized under multiplexed conditions to minimize cross-amplification or cross-hybridization resulting from possible sequence similarity between targets. 
     Mono- and 21-Fold Multiplexed Nested One-Step RT-PCRs with SAMRS-AEGIS Primers 
     These were carried out in 1× Reaction mix (Life Technologies) with RNA simulant (4 ng/μL) in a final volume of 20 μL accordingly to the Invitrogen protocol for the SuperScript One-Step RT-PCR with Platinum Taq (Life Technologies). The reaction mixture contained 0.2 mM of dZTP; 0.025 μM each of 21 pairs forward and Reverse hybrid SAMRS-AEGIS target-specific primers; 0.25 μM External AEGIS Forward and Reverse-biotinylated primers; 2.5 units RT/Platinum Taq enzyme Mix. Additional 1.5 mM MgSO 4  were added to the RT-PCR buffer. Cycling conditions were: one cycle of the cDNA synthesis and pre-denaturation (53° C. for 30 minutes and 94° C. for 2 minutes), 55 cycles of PCR (94° C. for 15 seconds, 53° C. for 30 seconds, and 70° C. for 30 seconds) and final extension at 72° C. for 5 minute. A “no-target” PCR negative control was included with each assay run. To favor incorporation of biotin-labelled reverse primers to maximize hybridization sensitivity, the second PCR was performed with only reverse biotinylated primer (reverse primer extension reaction, RPER). 
     Digestion of Excess Primers and dNTPs 
     To destroy excess primers and deactivate dNTPs prior to RPER, ExoSAP-IT enzyme mixture (2 μL, Affymetrix, Cleveland, Ohio USA) were added to aliquots (5 μL) of standard or SAMRS-AEGIS nested PCR. Reaction mixtures were incubated at 37° C. for 30 minutes and the enzyme mixture was destroyed by heating at 80° C. for 20 minutes. Treated PCR products were added directly to the Reverse Primer Extension reaction. 
     Reverse Primer Extension Reaction (RPER) 
     Briefly, a RPER (20 μL) was set up in 1× ThermoPol Buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1% Triton X-100, pH 8.8 at 25° C.) with 3 μL of each ExoSAP-treated RT-PCR product, 5′-biotinylated external (common) Reverse AEGIS primer (0.2 μM), and Vent (exo-) DNA polymerase (1 unit NEB). Without conversion (an “extension” reaction), dNTPs (final 0.2 mM each) were added. For the dZ incorporation into the final amplicon (“conversion”), nucleoside triphosphates (dATP, dTTP, dGTP, and dZTP, final concentration 0.2 mM of each) were added. The “extension” and “conversion” reaction mixtures were incubated in s BioRad (DNA Engine) Peltier Thermal Cycler at 95° C. for 1 min, followed by 20 cycles (94° C. for 20 seconds, 55° C. for 30 seconds, 72° C. for 30 seconds). A final incubation was run at 72° C. for 1 minute. Reaction mixtures were then held at 4° C. and quenched with 4 mM EDTA. 
     For standard RT-PCR products, a set of 21 reverse target-specific primers (0.2 μM each) was added to the “extension” or “conversion” reactions. The other reaction components were the same as above. 
     Probe Coupling to Beads 
     Capture probes modified with an amino-C12 linker at the 5′-end were coupled to Luminex MicroPlex carboxylated micro-spheres (“beads”) by a carbodiimide-based procedure according to the manufacturer&#39;s protocol. Briefly, for each combination of probe and bead set (Table 5), 2.5 million Luminex beads were resuspended in 0.1 M MES buffer (morpholine ethane sulfonic acid, 50 μL, pH 4.5) with probe (4 μL of 0.1 mM stock to give 0.4 nanomole final concentration), and treated twice with 1-ethyl-3-[3-dimethylamino-propyl]-carbodiimide hydrochloride (EDC, 5 μL of a 10 mg/mL solution, Thermo Scientific/Pierce, Rockford, Ill.) at room temperature for 30 min, rinsed in Tween 20 (0.02% aqueous solution), then rinsed with a sodium dodecylsulfate solution (0.1%), and resuspended in Tris-EDTA buffer (pH 8.0) to 5.0. 
     Luminex Direct Hybridization (DHA) 
     This was performed accordingly to the “no wash” Luminex protocol (http://www.luminexcorp.com/Support/SupportResources/). In a pilot experiment “wash” and “no wash” Luminex protocols were compared; no differences were found between two protocols when applied to our target-specific probes&#39; design. Briefly, aliquots (5 μL) of each extension or conversion reaction were transferred to 96-well plates (96-well PCR thermo polystyrene plates; Costar). Hybridization buffer (25 μL of 2×Tm 0.4 M NaCl; 0.2 M Tris, 0.16 Triton X-100, pH 8.0) containing 100 of each target-specific probe-coupled microsphere set per μL or totally 2,500 target-specific probe-coupled each microsphere types. Microspheres were vortexed and sonicated for 20 seconds. The total volume was adjusted to 50 μL by 20 μl of ddH 2 0. 25 μl of ddH 2 0 were added to each Background well (negative control). Hybridization was performed at 55° C. accordingly to the direct hybridization protocol (DHA) provided by Luminex: 95° C. for 5 min, cool to 55° C. at a speed of 0.1° C./second, 15 mM at the hybridization T 55° C. Tm buffer (25 μL of 1×) containing streptavidin-R-phycoerythrin (2 μg, PJRS14, PROzyme) were added to the each hybridization mixture, which was then incubated at 56° C. for 5 min. Hybridization reactions were carried out in triplicate, and “no-target” controls were run in replicates of 6. Beads were analyzed for internal bead color and R-phycoerythrin reporter fluorescence using a Luminex 200 analyzer (Luminex xMAP Technology, Luminex Corporation) and the xPonent Software solutions. The median reporter fluorescence intensity (MFI) was computed for each bead type in the sample. The instrument&#39;s gate setting was established before the samples were run, and was maintained throughout the course of the study. 
     Results 
     Twenty-one mosquito-borne arboviruses were selected (Table 1) to assemble and develop xMAP Luminex assays diagnostic panel based on PCR amplification and using the artificial SAMRS-AEGIS technology [Yan10; Yan13]. 
     Viral simulant RNAs were produced in vitro by transcription of the appropriate templates using T7 RNA polymerase. In pilot experiments, SuperScript One-Step RT-PCR with Platinum Taq (Life Technology) was found to be more sensitive and robust than other enzyme combinations tested, and thus able to support the nested PCR amplification with external primers containing the nonstandard P nucleotide, which pairs with the Z nucleotide. The target-specific standard or hybrid SAMRS-AEGIS forward and reverse primer pairs designed for the panel were tested first by monoplexed one-step RT-PCR with viral RNA simulants. Each monoplexed RT-PCR produced the expected amplicons, which were visualized by the ethidium-bromide staining following electrophoresis. Multiplexed RT-PCR conditions were established in a series of preliminary experiments (data not shown). Finally, multiplexed nested RT-PCRs were executed with each viral target using 21 pairs of specific SAMRS-AEGIS primers and AEGIS external primers (Table 2). PCR amplicons generated under optimized conditions were visualized on ethidium bromide gel as clearly resolved bands of the expected sizes ranging from 59 to 160 base pairs. The PCR negative control showed non-substantial level of primer-dimerization. 
     Amplicons containing only standard nucleotides were also produced by multiplexed one-step RT-PCRs and analyzed by agarose-gel electrophoresis. The 21-fold multiplexed reactions were primed with full set of target-specific primers, each at the final concentration of 0.4 The standard PCR amplicons were visualized on the ethidium bromide gel as clearly resolved bands of the expected sizes. 
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