Abstract:
The disclosed invention teaches processes to amplify oligonucleotides by contacting templates and primers with DNA polymerases and triphosphates of non-standard nucleotides, which form nucleobase pairs fitting the standard Watson-Crick geometry, but joined by hydrogen bonding patterns different from those that join standard A:T and G:C pairs. Thus, this invention relates to nucleotide analogs and their derivatives that, when incorporated into DNA and RNA, expand the number of replicatable nucleotides beyond the four found in standard DNA and RNA. The invention further relates to polymerases that incorporate those non-standard nucleotide analogs into oligonucleotide products using the corresponding triphosphate derivatives, and more specifically, polymerases and non-standard nucleoside triphosphates that support the polymerase chain reaction (PCR), including PCR where the products contain more than one non-standard nucleotide unit.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
     This application is the U.S. national stage application of International Patent Application No. PCT/US2009/003595, filed Jun. 16, 2009, which claims the benefit of U.S. Provisional Patent Application No. 61/132,225, filed Jun. 17. 2008, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     The United States Federal Government may have rights under this application. 
    
    
     THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT 
     Not applicable. 
     INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC 
     None 
     The Sequence Listing for this application is labeled “Seq-List.txt” which was created on Nov. 23,2010 and is 15 KB. The entire contents of the sequence listing is incorporated herein by reference in its entirety. 
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to processes that copy oligonucleotides in a polymerase chain reaction (PCR) where these oligonucleotides incorporate nucleotide analogs (“non-standard nucleotides”) that form base pairs joined by hydrogen bonding patterns not found in standard nucleotides A, T, G and C. The invention relates more specifically to processes that amplify oligonucleotides holding more than one non-standard nucleotides, including non-standard nucleotides at adjacent positions in the oligonucleotides chain, and amplification in a nested PCR format. 
     2. Description of Related Art 
     Natural oligonucleotides bind to complementary oligonucleotides according to well-known rules of nucleobase pairing first elaborated by Watson and Crick, where adenine (A) pairs with thymine (T) (or uracil, U, in RNA), and guanine (G) pairs with cytosine (C), with anti-parallel complementary strands. In this disclosure, “DNA”, “oligonucleotide”, or “nucleic acid” is understood to include DNA and RNA, as well as derivatives where the sugar is modified, as in 2′-O-methyl and 2′,3′-dideoxynucleoside derivatives, where the nucleobase has an appendage, and these nucleic acids and their analogs in non-linear topologies, including as dendrimers, comb-structures, and nanostructures, and analogs carrying appendages or tags (e.g., fluorescent, functionalized, or binding, such as biotin). Further, “polymerase” in this application is meant to include DNA polymerases of all families, RNA polymerases, and reverse transcriptases. 
     These pairing rules allow specific hybridization of oligonucleotides to complementary oligonucleotides, making oligonucleotides valuable as probes in the laboratory, in diagnostics, as messages that direct the synthesis of proteins, and in other applications known in the art. Such pairing is used, for example and without limitation, to capture oligonucleotides to beads, arrays, and other solid supports, allow nucleic acids to fold in hairpins, beacons, and catalysts, support function, such as fluorescence, quenching, binding/capture, and catalysis, and as part of complex structures, including dendrimers and nanostructures, and scaffolds to guide chemical reactions. 
     Further, base pairing underlies the enzymatic synthesis of oligonucleotides complementary to a template. Here, assembly of building blocks from nucleoside triphosphates is directed by a template to form a complementary oligonucleotide with a complementary sequence. This is the basis for replication in living systems, and underlies technologies for enzymatic synthesis and amplification of specific nucleic acids by enzymes such as DNA and RNA polymerase, the polymerase chain reaction (PCR), and assays involving synthesis, ligation, cleavage, immobilization and release, inter alia. 
     Watson-Crick pairing rules can be understood as the product of two rules of complementarity: (1) size complementarity (a big purine pairs with a small pyrimidine) and (2) hydrogen bonding complementarity (hydrogen bond donors pair with hydrogen bond acceptors). However, as noted by U.S. Pat. Nos. 5,432,272, 5,965,364, 6,001,983, 6,037,120, 6,140,496, 6,627,456, and 6,617,106, Watson-Crick geometry can accommodate as many as 12 nucleobases forming 6 mutually exclusive pairs. Of these, four nucleobases forming two pairs are designated “standard”, while eight nucleobases forming four pairs were termed “non-standard”, and may be part of an “artificially expanded genetic information system” (AEGIS). 
     To systematize the nomenclature for the hydrogen bonding patterns, the hydrogen bonding pattern implemented on a small component of a nucleobase pair are designated by the prefix “py”. Following this prefix is the order, from the major to the minor groove, of hydrogen bond acceptor (A) and donor (D) groups. Thus, both thymine and uracil implement the standard hydrogen bonding pattern pyADA. The standard nucleobase cytosine implements the standard hydrogen bonding pattern pyDAA. Hydrogen bonding patterns implemented on the large component of the nucleobase pair are designated by the prefix “pu”. Following the prefix, hydrogen bond donor and acceptor groups are designated, from major to minor groove, by “A” and “D”. Thus, the standard nucleobases adenine and guanine implement the standard hydrogen bonding patterns puDA- and puADD respectively. 
     A central teaching of this disclosure is that hydrogen-bonding patterns are distinct from the organic molecule that implemented them. Thus, guanosine implements the puADD hydrogen-bonding pattern. So does, however, 7-deazaguanosine, 3,7-dideazaguanosine, and many other purines and purine analogs, including those that carry side chains carrying functional groups, such as biotin, fluorescent, and quencher groups. Which organic molecule is chosen to implement a specific hydrogen-bonding pattern determines, in part, the utility of the non-standard hydrogen-bonding pattern, in various applications to which it might be applied. 
     Claims of U.S. Pat. No. 5,432,272 and its successors covered non-standard bases that implemented the pyDDA hydrogen bonding pattern. Subsequent efforts to use these, however, encountered problems, including epimerization [Voe96a,b], oxidation [Von95], and uncharacterized decomposition. Accordingly, Benner invented a new non-standard nucleoside, 6-amino-5-nitro-3-(1′-beta-D-2′-deoxyribofuranosyl)-2-(1H)-pyridone (dZ) to implement the pyDDA hydrogen bonding pattern. The nitro group rendered the otherwise electron-rich heterocycle stable against both oxidation and epimerization under standard conditions. When paired with a corresponding puAAD nucleotide, duplexes were formed with stabilities that, in many cases, were higher than those observed in comparable strands incorporating the dG:dC nucleobase pair [Yan06]. This invention is covered by U.S. patent application Ser. No. 11/372,400, which is incorporated herein by reference. Contents of this patent application have been published [Hut03]. 
     While Z supports binding of oligonucleotides containing it to complementary strands that match a nucleobase implementing the puAAD hydrogen bond pattern, it was not clear that polymerases would accept this unnatural base pair. Polymerases are known to be idiosyncratic [Hor95], meaning that experimentation is necessary to ascertain whether a specific implementation of a non-standard hydrogen bonding scheme can be accepted by a polymerase. 
     Thus, it was necessary to show by experiment that polymerases could incorporate dZ and dP. This was done for oligonucleotides containing a single dZ or dP [Yan07], which was published less than a year before the priority date of the instant application. However, [Yan07] showed that the dZ and dP are lost in multiple PCR cycles with Taq and Deep Vent (exo−) polymerases, perhaps via a mechanism where deprotonated dZ mispairs with dG (or deprotonated dG pairs with dZ), while protonated dC mispairs with dP (or protonated dP pairs with dC). Thus, this art teaches away from any use of the non-standard dZ:dP nucleobase pair in higher level PCR, defined as PCR that creates amplicons with multiple non-standard nucleotides. 
     BRIEF SUMMARY OF THE INVENTION 
     This invention concerns processes that amplify oligonucleotides containing non-standard nucleotides ( FIG. 1 ) in PCR where those oligonucleotides are not restricted in sequence to containing only a single nonstandard nucleotide, and where those oligonucleotides are not restricted in sequence to those where no adjacent nonstandard nucleotides appear, and amplification is done in a nested PCR format [Bro97]. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . A set of heterocycles implementing a set of hydrogen bonding patterns. The heterocycles used in the processes of the instant invention are labeled dZ and dP. 
         FIG. 2 . Schematic showing nested PCR, where nucleotides from an artificially expanded genetic information system (AEGIS) are included in the tags (vertically hashed lines) attached to chimeric PCR primers. Chimeric primers initiating the PCR may be at low concentrations, diminishing the possibility of off-target priming. After their consumption in early PCR cycles, PCR continues via priming with external primers. 
         FIG. 3 . Autoradiograph of an electrophoresis gel showing PCR products as described in Example 1. _Left: PCR using primers only standard nucleotides in both the amplicon binding region and in the 5′-tag. Right. PCR using primers containing dP in the 5′-tag. 
         FIG. 4 . Autoradiograph of an electrophoresis gel showing nested PCR products obtained as described in Example 2. 
         FIG. 5 . Autoradiograph of an electrophoresis gel showing primer extension products obtained as described in Example 3 from amplicons containing adjacent dZs. 
         FIG. 6 . Autoradiograph of an electrophoresis gel showing primer extension products obtained as described in Example 4. 
         FIG. 7 . Autoradiograph of an electrophoresis gel showing PCR products obtained as described in Example 5 
         FIG. 8 . Autoradiograph of an electrophoresis gel showing that nested PCR external primers containing AEGIS components produces cleaner PCR products (right) than with standard external primers (left). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     6-Amino-5-nitro-3-(1′-beta-D-2′-deoxyribofuranosyl)-2(1H)-pyridone (dZ), its protected phosphoramidite derivatives suitable for chemical oligonucleotides synthesis, its triphosphate and its thiotriphosphate are reported in U.S. patent application Ser. No. 11/372,400 and in [Hut03]; both are incorporated herein by reference. Its complement, 2-amino-8-(1′-β-D-2′-deoxyribofuranosyl)-imidazo[1,2-α]-1,3,5-triazin-4(8H)-one (implementing puAAD, as dP) and these derivative are reported in [Hut03][Yan06], the synthetic procedures therein being incorporated herein by reference. These are preferred as non-standard nucleotides in the instant invention, although any non-standard nucleotide shown in  FIG. 1  might be used in an eternal primer in a PCR. 
     It is known in art published less than a year before the priority date of the instant application [Yan07] that PCR amplification is possible for oligonucleotides that contain a single dP or a single dZ in the amplicon. However, the prior art shows that upon multiple PCR cycles, dP and dZ are removed from the amplicon. The instant invention was made following first the recognition it was possible to place dP and/or dZ in the PCR primers themselves. To amplify standard oligonucleotides, the dP and dZ would not be in the region of the primer that contacts that target, but rather within an oligonucleotide tag (or tail) appended to the 5′-end of the PCR primer that does contact the standard oligonucleotide target. PCR primers having two parts, a 5′-end containing dP and/or dZ (as well as standard nucleotides, optionally) and a 3′-end that is complementary to the target, are called “chimeric primers”. PCR amplification with chimeric primers is then done in a nested fashion [Bro97]. Any loss of the non-standard nucleotide is restored in a subsequence PCR cycle, solving the loss problem described in the literature. After the chimeric primers are consumed in the first cycles, “external primers” having the sequence of only the 5′-tags continue the PCR. 
     Not known in any art published before the priority date of the instant application, and not anticipatable from the prior art given the idiosyncrasies of polymerases when challenged with unnatural nucleoside triphosphate substrates [Hor95], it was then discovered that PCR could succeed with certain polymerases even for amplicons that contain multiple dPs and/or multiple dZs. Further, we disclose here for the first time that certain polymerases support incorporation of adjacent dZs and adjacent dPs in prime extension reactions 
     A third discovery was also unanticipated by us or the art. It turned out that by doing nested PCR with dZ and/or dP in the external primers, it was possible to obtain cleaner PCR products. Given this result, it became evident that PCR with dZ and dP in the primers was a useful, patentable invention, as is herein claimed. 
     Accordingly, the invention together with its associated discoveries provides a process for amplifying an oligonucleotide sequence in a PCR format, where a forward chimeric primer and a reverse chimeric primer are contacted with that target sequence. The forward chimeric primer is complementary to a region at the 3′-end of said sequence (as in any PCR), the reverse chimeric primer is identical in sequence to a region at the 5′-end of said sequence (as in any PCR), and the forward and reverse primers are joined at their 5′-prime ends to oligonucleotide tags having independently selected sequences that contain at least one non-standard nucleotide (see  FIG. 1 ), where the presently preferred non-standard nucleotide is dZ or dP, most preferably dP. Then, as with standard OCR, the mixture is incubated the mixture with a DNA polymerase, RNA polymerase, or a reverse transcriptase, depending on the nature of the oligonucleotide to be amplified, together with the triphosphates needed to complement the nucleotides in the primers and sequence. The presently preferred polymerases are Phusion and Vent or Deep Vent having exonuclease activity, as described further in the examples. This process, of course, comprises a simpler PCR process that amplifies an oligonucleotide sequence that contains one or more non-standard nucleotides, preferably dZ or dP. PCR amplification of an oligonucleotide containing just one was disclosed less than a year before the priority date in [Yan07], and therefore is patentable under United States law. PCR amplification of oligonucleotides containing more than one non-standard nucleotide and adjacent non-standard nucleotides was not in the prior art prior to the priority date, and is not rendered obvious by [Tan07], and is therefore internationally patentable. 
     EXAMPLES 
     Example 1 
     PCR with dZ and dP 
     This example demonstrates that chimeric primers containing dZ and dP in their external segments support PCR. The following oligonucleotides were prepared by phosphoramidite synthesis. These include two reverse chimeric primers, identical except that in R-36-Nest, some of the G&#39;s were replaced by P&#39;s in the segment not complementary to the template: 
                             R-36-Std:           SEQ ID 1           3′- CCATGGTAGCTATGCGCAA CGCTAGCGAGGAAGGAC-5′-P 32                         R-36-Nest:           SEQ ID 2           3′- CCATGGTAGCTATGCGCAA CPCTAPCGAPGAAPGAC-5′-P 32              
a pair of complementary template sequences:
 
                     Temp-R-47:       SEQ ID 3       5′-CCATGGGAGACCGCGGTGGGCCCGGCCGGGTACCATCGATACGCG       TT-3′               Temp-F-47:       SEQ ID 4       3′-GGTACCCTCTGGCGCCACCCGGGCCGGCCCATGGTAGCTATGCGC       AA-5′            
and two forward chimeric primers, one with the same replacements:
 
     
       
         
               
             
               
               
             
           
               
                 (F-34-Nest) 
               
             
          
           
               
                 5′-CTAPGACPACGPACTPC CCATGGGAGACCGCGGT -3′ 
                 SEQ ID 5 
               
               
                   
               
               
                 (F-34-Std) 
                   
               
               
                 5′-CTAGGACGACGGACTGC CCATGGGAGACCGCGGT -3′ 
                 SEQ ID 6 
               
             
          
         
       
     
     The underlined portions of the primers are complementary to the template. The portions not underlined are the tags that contain non-standard nucleotides. In separate experiments the chimeric and non chimeric primers were incubated under the following conditions: 
     
       
         
               
               
               
             
               
               
               
               
             
               
               
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                   
                   
                 Final 
               
               
                 Components 
                 Volume (μl) 
                 Concentration 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Nuclease-Free Water 
                 1.6 
                   
                   
               
               
                 (final volume of 20 μl) 
                   
                   
                   
               
               
                 Forward Primer: F-34-Nest (2 pmol/μl) 
                 5 
                 500 
                 nM 
               
               
                 Reverse Primer: R-36-Nest (1 pmol/μl, 
                 1 
                 50 
                 nM 
               
               
                 radiolabeled) 
                   
                   
                   
               
               
                 Reverse Primer: R-36-Nest 
                 6 
                 450 
                 nM 
               
               
                 (1.5 pmol/μl) 
                   
                   
                   
               
             
          
           
               
                 Template: 
                 Temp-F-47(1 pmol/μl) 
                 0.1 for each 
                 5 
                 nM 
               
               
                   
                 Temp-R-47(1 pmol/μl) 
                   
                   
                   
               
             
          
           
               
                 10 × Reaction Buffer 
                 2 
                 1× 
                   
               
               
                 dNTP (2 mM of each dNTP) 
                 2 
                 0.1 
                 mM each 
               
               
                 dZTP (2 mM) or Water (for negative 
                 2 
                 0.1 
                 mM 
               
               
                 control) 
                   
                   
                   
               
               
                 Taq (5 U/μl) 
                 0.2 
                 0.05 
                 U/μl 
               
               
                   
               
             
          
         
       
     
     The products were resolved by gel electrophoresis ( FIG. 3 ). These results show that primers containing multiple dP&#39;s support PCR works. The experiment does not show, however, that primers containing consecutive dPs effectively support PCR. 
     Example 2 
     Nested PCR 
     This experiment demonstrated the use of dZ and dP pairing in the external segments of a nested PCR [Bro97], shown schematically in  FIG. 2 . The following oligonucleotides were prepared by phosphoramidite synthesis. These were set up in three set of nested PCR experiments. The first used external primers, one containing dP, the other not: 
                                           F-17-Nest                  32 P-5′-CTAPGACPACGPACTPC-3′   SEQ ID 7                       F-17-Std                 32 P-5′-CTAGGACGACGGACTGC-3′   SEQ ID 8            
applied in a direct PCR experiment for a longer template that included Temp-R-47 in its middle:
 
                     Temp-R-81:       SEQ ID 9       5′-CTAGGACGACGGACTGCCCATGGGAGACCGCGGT GGGCC C GGCCG                 GGTACCATCGATACGCGTTGCGATCGCTCCTTCCTG-3′            
and two reverse external primers, one containing dP, the other not:
 
     
       
         
               
               
             
               
               
               
             
           
               
                   
                 R-17-Std: 
               
             
          
           
               
                   
                 3′-CGCTAGCGAGGAAGGAC-5′ 
                 SEQ ID 9 
               
               
                   
                   
               
               
                   
                 R-17-Nest: 
                   
               
               
                   
                 3′-CPCTAPCGAPGAAPGAC-5′ 
                 SEQ ID 10 
               
             
          
         
       
     
     These were incubated using the following procedure in Experiment A. 
     
       
         
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                   
                 Volume 
                 Final 
               
               
                 Components 
                 (μl) 
                 Concentration 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Nuclease-Free Water 
                 7.65 
                   
                   
               
               
                 (final volume of 20 μl) 
                   
                   
                   
               
               
                 Forward Primer: F-17-Std or Nest 
                 2.25 
                 225 
                 nM 
               
               
                 (2 pmol/μl) 
                   
                   
                   
               
               
                 Forward Primer: F-17-Std or Nest 
                 0.5 
                 25 
                 nM 
               
               
                 (1 pmol/μl, radiolabeled) 
                   
                   
                   
               
               
                 Reverse Primer: R-17-Std or Nest 
                 2.5 
                 250 
                 nM 
               
               
                 (2 pmol/μl) 
                   
                   
                   
               
               
                 Template: Temp-R-81 (0.01 pmol/μl) 
                 0.5 
                 0.25 
                 nM 
               
             
          
           
               
                 10 × Reaction Buffer (MgCl 2  (15 mM)) 
                 2 
                 1 × (MgCl 2  (1.5 mM)) 
               
               
                 MgCl 2  (25 mM) 
                 0.4 
                 MgCl 2  (0.5 mM) 
               
             
          
           
               
                 dNTP (2 mM of each dNTP) 
                 2 
                 0.2 
                 mM each 
               
               
                 dZTP (2 mM) 
                 2 
                 0.2 
                 mM 
               
               
                 Taq (5 U/μl) 
                 0.2 
                 0.05 
                 U/μl 
               
               
                   
               
             
          
         
       
     
     The standard primers F-17-Std and R-17-Std should amplify the Temp-R-81 target, leading to a band in a gel electrophoresis resolution that migrates as an 81-mer. This is in fact the case ( FIG. 4 , A-Std lane). If dP does not bind to dC, then the AEGIS dP-containing primers should not yield and 81-mer band. This is also the case ( FIG. 4 , A-AEGIS). 
     In another experiment, the following recipe was used in a nested PCR experiment: 
     
       
         
               
               
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                   
                 Volume 
                   
               
               
                 Components 
                 (μl) 
                 Final Concentration 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Nuclease-Free Water 
                 6.65 
                   
                   
               
               
                 (final volume of 20 μl) 
                   
                   
                   
               
               
                 Forward Primer: F-17-Std or Nest 
                 2.25 
                 225 
                 nM 
               
               
                 (2 pmol/μl) 
                   
                   
                   
               
               
                 Forward Primer: F-17-Std or Nest 
                 0.5 
                 25 
                 nM 
               
               
                 (1 pmol/μl, radio-labeled) 
                   
                   
                   
               
               
                 Reverse Primer: R-17-Std or Nest 
                 2.5 
                 250 
                 nM 
               
               
                 (2 pmol/μl) 
                   
                   
                   
               
               
                 Template: Temp-R-47(0.01 pmol/μl) 
                 0.5 
                 0.25 
                 nM 
               
               
                 F-34-Std or F-34-Nest (0.1 pmol/μl) 
                 0.5 
                 2.5 
                 nM 
               
               
                 R-36-Std or R-36-Nest (0.1 pmol/μl) 
                 0.5 
                 2.5 
                 nM 
               
             
          
           
               
                 10 × Reaction Buffer (MgCl 2  (15 mM)) 
                 2 
                 1 × (MgCl 2  (1.5 mM)) 
               
               
                 MgCl 2  (25 mM) 
                 0.4 
                 MgCl 2  (0.5 mM) 
               
             
          
           
               
                 dNTP (2 mM of each dNTP) 
                 2 
                 0.2 
                 mM each 
               
               
                 dZTP (2 mM) 
                 2 
                 0.2 
                 mM 
               
               
                 Taq (5 U/μl) 
                 0.2 
                 0.05 
                 U/μl 
               
               
                   
               
               
                 Note: 
               
               
                 1 × standard Taq Reaction Buffer (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl 2 , pH 8.3 at 25° C.). 
               
               
                 1 × ThermoPol Reaction Buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 , 0.1% Tritonx-100, pH 8.8 at 25° C.). 
               
             
          
         
       
     
     Experiment B used F-34-Std and R-36-Std as chimeric primers. These should generate products when amplified with external primers built without dP, but not when amplification was sought with external primers containing dP. This was in fact the case ( FIG. 4 , B-Std and B-AEGIS, respectively). Experiment C used F-34-Nest and R-36-Nest as chimeric primers. These should not generate products when amplified with external primers built with dP, but should generate when amplification was sought with external primers containing dP. This was in fact the case ( FIG. 4 , C-Std and C-AEGIS, respectively). These experiments shows showed the ability of DNA polymerase to support a six letter PCR with dA, dT, dG, dC, dZ, and dP as the six letters. They also demonstrate the orthogonality of the process. Nested PCR works with AEGIS external primers when it should and not when it should not, and vice versa. 
     Example 3 
     Primer Extension Through Adjacent dZs in a Template 
     Given the well-known idiosyncrasies of polymerases and the possibility of strong neighbor effects [Hor95], it was not clear that these results would be extendable to PCR amplifications where dZ or dP are adjacent in a template, requiring the incorporation of dP and dZ adjacent in the template. The following experiments were done to screen thermophilic polymerases for their ability to incorporate dPTP opposite dZ in the template. This was done at the following concentrations: [dATP]=[dCTP]=[dTTP]=100 microM), dGTP (5 microM to 100 microM), or dPTP (5 microM to 100 microM) at pH7.0 or 7.5, with the following oligonucleotides (R-19-S was P-32 labeled at its 5′-end): 
     
       
         
               
               
             
           
               
                   
                 R-19-S: 
               
               
                   
                 SEQ ID 11 
               
               
                   
                 5′-GGTACCATCGATACGCGTT-3′ 
               
               
                   
                   
               
               
                   
                 R-36-Nest-6Z: 
               
               
                   
                 SEQ ID 12 
               
               
                   
                 3′-CCATGGTAGCTATGCGCAAGTZZTTZZTCGZTAGZG-5′ 
               
             
          
         
       
     
     5′- 32 P-labeled primer R-19-S (2 pmole, final assay concentration 50 nM) was annealed to a template sequence R-36-Nest-6Z (3 pmole, final assay concentration 75 nM) by heating (5 min 95° C.) and then slow cooling (0.5 h) to room temperature. dATP, dTTP and dCTP (4 nmole each, final 100 microM) and dGTP (final 10 microM), or dPTP (final 10 microM) were added at room temperature. The reaction mixture was pre-incubated at 72° C. for 30 seconds and followed by the addition of Taq DNA polymerase to give a final volume of 40 microL. The mixture was incubated at 72° C. for 4 minutes, and quenched by dilution into PAGE loading/quench buffer (10 microL, 10 mM EDTA in formamide). Samples were resolved by electrophoresis using a 14% PAGE (7 M urea). The gel was analyzed using MolecularImager software. 
     These results ( FIG. 5 ) showed that Vent and Deep Vent performed better than Taq. Without wishing to be bound by theory, this may be due to their exonuclease activities. 
     Example 4 
     Incorporation of dZTP Opposite Consecutive Template dPs 
     To compare the efficiency and fidelity of DNA polymerases (Taq, Vent (exo+), and DV (exo+)) incorporating dZTP opposite two consecutive dPs in a template, 5′- 32 P-labeled primer T7-Y-RS-S16 (0.2 pmole of hot primer plus 4 pmole of cold primer, final assay concentration 70 nM) was annealed to template T7-PP-Temp (6 pmole, final assay concentration 100 nM) in 1×. Thermopol polymerase reaction 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=8.0 at room temperature) by heating (5 min at 95° C.) and then slow cooling (0.5 h) to room temperature. dNTP (each final 0.1 mM) and dZTP (final 0.1 mM, with (+) or without (−)) were added at room temperature. The reaction mixture was cooled to 4° C. for 1 min and followed by the addition of Taq (2.5 units), Vent (exo+), or Deep Vent (exo+) DNA polymerase (2 units for Vent and Deep Vent) to give a final volume of 60 microL. The primer was extended at 65° C. and aliquots (7 microL) were taken from each reaction at time intervals (1, 2, 4, 8, and 16 min), quenched by PAGE loading/quench buffer (7 microL, 10 mM EDTA in formamide) and resolved by electrophoresis using a 16% PAGE (7 M urea). The gel was analyzed using MolecularImager. These oligonucleotides were used: 
     
       
         
               
             
           
               
                 Negative control (−): dNTP (each 0.1 mM) 
               
               
                 T7-Y-RS-S16: 
               
               
                 SEQ ID 13 
               
               
                 3′-GAAAT*CACTCCCAATTAAGCG-5′ 
               
               
                   
               
               
                 T7-PP-Temp: 
               
               
                 SEQ ID 14 
               
               
                 5′-GCGTAATACGACTCACTATAGACGAPPCTACTTTAGTGAGGGTTA 
               
               
                 ATTCGC-3′ 
               
               
                   
               
               
                 Positive control (+): dNTP (each 0.1 mM), and 
               
               
                 dZTP (0.1 mM) 
               
               
                 T7-Y-RS-S16: 
               
               
                 SEQ ID 15 
               
               
                 3′-GAAAT*CACTCCCAATTAAGCG-5′ 
               
               
                   
               
               
                 T7-PP-Temp: 
               
               
                 SEQ ID 16 
               
               
                 5′-GCGTAATACGACTCACTATAGACGAPPCTACTTTAGTGAGGGTTA 
               
               
                 ATTCGC-3′ 
               
             
          
         
       
     
     The order of performance of the polymerases tested is Deep Vent (exo+)&gt;Vent (exo+)&gt;Taq. In the absence of dZTP, Deep Vent and Vent misincorporates only one dCTP opposite the first dP. However, Taq can misincorporate dCTP opposite two consecutive dPs, and then keep extending primer. All are better than exo(−) polymerases (not reported in [Yan07]). 
     Example 5 
     PCR with Amplicons Containing Multiple Adjacent dPs and dZs 
     To compare the outcome of PCR with templates containing one or two adjacent dPs, the following oligonucleotides were prepared: 
     
       
         
               
             
           
               
                 T7-Z-RS-S16: 
               
               
                 SEQ ID 17 
               
               
                 5′-GCGTAATACGACTCAC*TATAG-3′ 
               
               
                 (Template-A) 
               
               
                   
               
               
                 T7-G-51-Std: 
               
               
                 SEQ ID 18 
               
               
                 5′-GCGTAATACGACTCACTATAGACGAGCGTACTTTAGTGAGGGTTA 
               
               
                 ATTCGC-3′ 
               
               
                 (Template-B) 
               
               
                   
               
               
                 T7-P-Temp: 
               
               
                 SEQ ID 19 
               
               
                 5′-GCGTAATACGACTCACTATAGACGAPCGTACTTTAGTGAGGGTTA 
               
               
                 ATTCGC-5′ 
               
               
                 (Template-C) 
               
               
                   
               
               
                 T7-PP-Temp: 
               
               
                 SEQ ID 20 
               
               
                 5′-GCGTAATACGACTCACTATAGACGAPPCTACTTTAGTGAGGGTTA 
               
               
                 ATTCGC-3′ 
               
               
                   
               
               
                 T7-Y-RS-S16: 
               
               
                 SEQ ID 21 
               
               
                 3′-GAAAT*CACTCCCAATTAAGCG-5′ 
               
             
          
         
       
     
     These were incubated under the following conditions: 
     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                   
                 Volume 
                   
               
               
                 Components 
                 (μl) 
                 Final Concentration 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Nuclease-Free Water 
                 17 
                   
                   
               
               
                 (final volume of 40 μl) 
                   
                   
                   
               
               
                 Forward Primer: T7-Z-RS-S16 
                 1 
                 0.25 
                 μM 
               
               
                 (10 pmol/μl) 
                   
                   
                   
               
               
                 Reverse Primer: T7-Y-RS-S16 
                 1 
                 0.25 
                 μM 
               
               
                 (10 pmol/μl) 
                   
                   
                   
               
               
                 Template: Three different Templates 
                 1 + 4 (H2O) 
                 0.25 
                 nM 
               
               
                 (A, B, and C) (0.01 pmol/μl) 
                   
                   
                   
               
               
                 10 × Thermopol Buffer (pH = 8.0) 
                 4 
                   
                   
               
               
                 dNTP (2 mM) 
                 4 
                 0.2 
                 mM each 
               
               
                 dZTP (2 mM) 
                 4 
                 0.2 
                 mM 
               
               
                 dPTP (2 mM) 
                 4 
                 0.2 
                 mM 
               
               
                 Hot Start Taq (5 U/μl) 
                 0.5 
                 0.06 
                 U/μl 
               
               
                   
               
               
                 ThermoPol Reaction 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 8.0 at 25° C.). 
               
               
                 PCR: one cycle of 95° C. for 15 min; 26 cycles of 95° C. for 20 s, (55° C. for 30 s, 72° C. for 1 min or 2 min; 72° C. for 5 min; then stored at 4° C. 
               
             
          
         
       
     
     The results are shown in  FIG. 7 . All PCRs generates some degree of 51-mer product. Template A contained only standard nucleotides. Template B contained a single dP. Template C contained a series of dPs, including two adjacent dPs. 
     Example 6 
     Prevention of Primer Dimerization with AEGIS Containing Primers 
     To demonstrate that if dP or dZ were incorporated into PCR primers in place of one or more dGs or dCs, then the synthetic primers containing dPs or dZs would not find their perfectly matched complementary strands in a primer pool, a 17-mer, 5′-CAGGAAGGAGCGATCGC-3′ (SEQ ID 35) was deliberately designed to form a self-dimer with 8 base pairs at the 3′-end (underline region, T m =32° C.), and subjected to PCR conditions. As expected, primer-dimer formed rapidly. In contrast, perfectly mismatched primers 5′-CAGGAAGGAGCPATCPC-3′ (SEQ ID 36) and 5′-CAGGAAGGAGZGATZGC-3′ (SEQ ID 37), which would form primer-dimers only by mismatching dP with dC (in the first case) and dZ with dG (in the second) gave no detectable amplicon under the same conditions, even after 45 cycles. 
     Example 7 
     Preferred DNA Polymerases and Optimized Amplification Conditions 
     With several polymerases able to replicate efficiently DNA fragments containing multiple dPs and dZs, the preferred polymerase having the highest PCR efficiency to amplify target using a nested PCR architecture with AEGIS nucleotides in the external primers was determined. Taq, 9° N, Deep Vent (both exo +  and exo − ), Vent (both exo +  and exo − ), Phusion, and Herculase were tested; the PCR efficiency was monitored by real-time PCR. The polymerase has the higher PCR efficiency generates more PCR amplicon and producing higher fluorescence signal at an earlier cycle of the PCR. Phusion was found to have the highest PCR efficiency among the polymerases tested with proofreading activity; Deep Vent (exo − ) is the most efficient among all the polymerases without exonuclease activity. For all polymerases tested, dP-containing nested PCR, in general, has higher PCR efficiency than that of the dZ-containing nested PCR. 
     Phusion DNA polymerase generates long templates with an accuracy and speed previously unattainable with a single enzyme. In addition, the error rate of Phusion is 50-fold lower than that of Taq, and about 6-fold lower than that of Vent and Deep Vent. Therefore, Phusion DNA polymerase was further optimized as a presently preferred polymerase for nested PCR with dP-containing external primers. The major infidelity during the 6-nucleotide PCR arises from misincorporation of dGTP opposite template dZs or dZTP opposite template dGs. This infidelity is pH dependent, when the pH of the buffer is low, the rate of misincorporation decreases. 
     To determine a preferred pH for PCR efficiency and fidelity, three types of nested PCR were conducted with Phusion HF buffer at four different pH values (7.0, 7.5, 8.0, and 8.5). Amplification was monitored by the real-time PCR with SYBR Green. For “type A” nested PCR, standard external and chimeric primers and four standard nucleotide triphosphates (dNTPs) were used; amplification curves in real-time show that PCR efficiency increases when the pH of the buffer decreases. After 30 cycles, the melting temperature of each PCR amplicon was measured and the size of each amplicon was analyzed by agarose (3%) gel. The T m  of each PCR amplicon generated under four different pH values is roughly the same (about 91.49±0.5° C.). 
     The type B nested PCR is identical to the type A nested PCR, except that dZTP was also included into the reaction. By comparing the melting temperature of each PCR amplicon in the type B reaction with that of the type A reaction, misincorporation of dZTP at different pHs was measured. The T m  of the amplicon improves as the pH of the reaction buffer decreases. For example, at the highest pH value tested (8.5), the T m  of the PCR amplicon is 3.75±0.05° C. below than that of the control PCR (type A); at the lowest pH value (7.0), the ΔT m  was to 0.29±0.05° C. below the fully standard PCR. For the type C nested PCR, dP-containing primers were used instead of standard primers, and PCR amplifications were conducted under the same conditions as type B nested PCR. The T m  of each PCR amplicon increases as the pH value of the reaction buffer decreases; the effect of pH on PCR efficiency and misincorporation of dZTP opposite template dG agreed with that with type B nested PCR. However, the T m  of each PCR amplicon in the type C nested PCR is higher (about 3.6° C.) than in type B nested PCR, this enhancement of the T m  is mainly due to the higher thermostability of the Z:P base pairs in the PCR amplicon. 
     The PCR amplicons obtained at four different pHs in type B nested PCR were cloned, and their sequences were verified by Sanger sequencing. This shows that misincorporation of dZTP opposite template G is insignificant and does not prevent the PCR amplicon of interest to be cloned and sequenced using the conventional Sanger method. This too was not expected given [Yan07], and can be used as a restrictive element of a claim. 
     Example 8 
     Nested PCR with AEGIS External Primers Cleans Up Multiplexed PCR 
     To show whether the dP-containing nested PCR can enhance the capability of multiplexed PCR, this system was applied to human genomic DNA, targeting the three genes associated with cancer: TOP1, HBEGF, and MYC. The oligonucleotides used in this experiment were: 
     
       
         
               
             
           
               
                 Top-F-External 
               
               
                 SEQ ID 22 
               
               
                 5′-TPTAPATTTPTATPTATPTATPAT-3′ 
               
               
                   
               
               
                 Top-F-Chimeric 
               
               
                 SEQ ID 23 
               
               
                 5′-TPTAPATTTPTATPTATPTATPATGACAGCCCCGGATGAGAAC-3′ 
               
               
                   
               
               
                 TOP-R-Chimeric 
               
               
                 SEQ ID 24 
               
               
                 3′-GTTAGCTCGACAACGTTAAGAACAPAGGPAAATPACTCPCA-5′ 
               
               
                   
               
               
                 Universal-R-4P 
               
               
                 SEQ ID 25 
               
               
                 3′-CAPAGGPAAATPACTCPCA-5′ 
               
               
                   
               
               
                 HBE-F-External 
               
               
                 SEQ ID 26 
               
               
                 5′-AAAPTATAPTAAPATPTATAPTAG-3′ 
               
               
                   
               
               
                 HBE-F-Chimeric 
               
               
                 SEQ ID 27 
               
               
                 5′-AAAPTATAPTAAPATPTATAPTAGCCCCAGTTGCCGTCTAGGA-3′ 
               
               
                   
               
               
                 HBE-R-Chimeric 
               
               
                 SEQ ID 28 
               
               
                 3′-TTCACGGTTTGTCTCATACAGGCCAPAGGPAAATPACTCPCA-5′ 
               
               
                   
               
               
                 Universal-R-4P 
               
               
                 SEQ ID 29 
               
               
                 3′-CAPAGGPAAATPACTCPCA-5′ 
               
               
                   
               
               
                 MYC-F-External 
               
               
                 SEQ ID 30 
               
               
                 5′-GTATTTPAPTAAPTAATTPATTPA-3′  
               
               
                   
               
               
                 MYC-F-Chimeric 
               
               
                 SEQ ID 31 
               
               
                 5′-GTATTTPAPTAAPTAATTPATTPATCCTCCTTATGCCTCTATC 
               
               
                 AT-3′ 
               
               
                   
               
               
                 MYC-R-Chimeric 
               
               
                 SEQ ID 32 
               
               
                 3′-CCTGAGAACTAGTTTCGCGCCCAPAGGPAAATPACTCPCA-5′ 
               
               
                   
               
               
                 Universal-R-4P 
               
               
                 SEQ ID 33 
               
               
                 3′-CAPAGGPAAATPACTCPCA-5′ 
               
             
          
         
       
     
     The external primers were adopted from Luminex&#39;s 5′-universal tag sequences, which were designed by the company to have unique sequences to avoid cross-hybridization and have roughly equal melting temperatures. To design the chimeric primers, universal external primer was added to either forward or reverse gene-specific primers, the other three external primers were also attached to gene-specific primers, the combination of each external primer to a certain gene-specific primer was optimized using primer design software (OligoAnalyzer 3.1), and following the general principles of multiplex PCR primer design to avoid cross-hybridization and hairpin structure of primers. 
     Three cancer genes in human genomic DNA were multiplexed amplified by standard nested PCR and dP-containing nested PCR using Phusion under seven different annealing temperatures. As shown in  FIG. 8 , standard nested PCR with all annealing temperatures give messy PCR results (left): at the lower annealing temperatures (from 53.4° C. to 58.8° C.), significant amounts of primer dimer (about 40 nucleotides in length) were generated; at the higher annealing temperatures (from 61.5° C. to 65.5° C.), non-specific PCR artifacts (PCR amplicons longer than the desired length) were produced along with the disappearance of the primer dimer. 
     However, the dP-containing nested PCR generated the desired PCR amplicons with minimal PCR artifacts (right). For the two control reactions (without genomic DNA), control A (standard nested PCR) gave some primer dimers (a 40-mer amplicon formed by standard external primers) and significant amount of long PCR amplicons, which may caused by the further priming from the primer dimer. The control B (dP-containing nested PCR) gave one band, which was formed by the dimerization of the dP-containing chimeric primers, as the 3′-ends of the chimeric primers are the standard gene-specific oligonucleotides. This dimerization could be further eliminated by reducing the concentration of the dP-containing chimeric primers. We further verified that the dP-containing multiplexed nested PCR also performed better than the standard nested PCR under HF Phusion buffer with other different pH values (8.5, 8.0, and 7.5). 
     This result was entirely unanticipated. Nested PCR using AEGIS external primers leads to cleaner multiplexed PCR. Without wishing to be bound by theory, this may arise because even with standard primers not having exact matched in a genome, standard primers have sufficient mismatches to prime at off-target sites. 
     LITERATURE CITED 
     
         
         [Bro97] Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., Little, S. (1997).  Nucleic Acids Res.  25, 3235-3241 
         [Hor95] Horlacher, J., Hottiger, M., Podust, V. N., Hübscher, U., Benner, S. A. (1995)  Proc. Natl. Acad. Sci.,  92, 6329-6333 
         [Hut03] Hutter, D. and Benner, S. A. (2003)  J. Org. Chem.,  68, 9839-9842 
         [Swi89] Switzer, C. Y., Moroney, S. E., Benner, S. A. (1989) Enzymatic incorporation of a new base pair into DNA and RNA.  J. Am. Chem. Soc.  111, 8322-8323 
         [Voe96a] Voegel, J. J., Benner, S. A. (1996)  Helv. Chim. Acta  79, 1863-1880 
         [Voe96b] Voegel, J. J., Benner, S. A. (1996)  Helv. Chim. Acta  79, 1881-1898 
         [Voe93] Voegel, J. J., von Krosigk, U., Benner, S. A. (1993) Synthesis and tautomeric equilibrium of 6-amino-5-benzyl-3-methylpyrazin-2-one. An acceptor-donor-donor nucleoside base analog.  J. Org. Chem.  58, 7542-7547 
         [Von95] von Krosigk, U., Benner, S. A. (1995)  J. Am. Chem. Soc.  117, 5361-5362 
         [Yan06] Yang, Z., Hutter, D., Sheng, P., Sismour, A. M. and Benner, S. A. (2006)  Nucleic Acids Res.,  34, 6095-6101. 
         [Yan07] Yang, Z., Sismour, A. M., Sheng, P., Puskar, N. L., Benner, S. A. (2007)  Nucl. Acids Res.  35, 4238-4249