Universal Tags With Non-Natural Nucleobases

Abstract

The present invention relates to amplification primers with a universal tag and sequencing primers comprising at least one non-natural nucleobase capable of hybridizing to a complementary non-natural nucleobase. The present invention further relates to amplification methods of nucleic acid amplification and sequencing using an amplification primer with a universal tag and sequencing primers, as well as kits and solid supports comprising such primers and tags.

1 . An oligonucleotide primer comprising:
 (a) a 3′ segment comprising a target-specific nucleotide sequence;   (b) a 5′ segment comprising a non-target-specific universal sequencing tag comprising at least one non-natural nucleobase capable of hybridizing to a complementary non-natural nucleobase and capable of being replicated during amplification.   
     
     
         2 . The oligonucleotide primer according to  claim 1 , wherein the at least one non-natural nucleobase of the universal sequencing tag is capable of hybridizing to a complementary non-natural nucleobase with a higher affinity relative to the natural nucleobase pairs G-C and A-T. 
     
     
         3 . The oligonucleotide primer according to  claim 1 , wherein the 5′ universal sequencing tag comprises a plurality of non-natural nucleobases capable of hybridizing to complementary non-natural nucleobases. 
     
     
         4 . The oligonucleotide primer according to  claim 1 , wherein the 5′ universal sequencing tag comprises a plurality of contiguous non-natural nucleobases capable of hybridizing to complementary non-natural nucleobases. 
     
     
         5 . The oligonucleotide primer according to  claim 1 , wherein the 5′ universal sequencing tag comprises at least three contiguous non-natural nucleobases capable of hybridizing to complementary non-natural nucleobases. 
     
     
         6 . The oligonucleotide primer according to  claim 1 , wherein the 5′ universal sequencing tag comprises a plurality of contiguous non-natural nucleobases capable of hybridizing to complementary non-natural nucleobases, and wherein the contiguous non-natural nucleobases are contiguous to the target-specific nucleotide sequence. 
     
     
         7 . The oligonucleotide primer according to  claim 1 , wherein the 5′ universal sequencing tag comprises a plurality of contiguous non-natural nucleobases capable of hybridizing to complementary non-natural nucleobases, and wherein the contiguous non-natural nucleobases are positioned 5′ of at least a portion of the target-specific nucleotide sequence. 
     
     
         8 . The oligonucleotide primer according to  claim 1 , wherein the non-natural nucleobases are selected from the group consisting of non-Watson-Crick complementary nucleobase analogs. 
     
     
         9 . The oligonucleotide primer according to  claim 1 , wherein the non-natural nucleobases are selected from the group consisting of Watson-Crick complementary nucleobase analogs having hydrogen bonding interactions that can be discriminated from natural nucleobase pairs. 
     
     
         10 . The oligonucleotide primer according to  claim 1 , wherein the plurality of non-natural nucleobases of the amplification primer are independently selected from the group consisting of isocytosine, isoguanine, and 5-methylisocytosine. 
     
     
         11 . The oligonucleotide primer according to  claim 1 , wherein at least one non-natural nucleobase is isocytosine. 
     
     
         12 . The oligonucleotide primer according to  claim 1 , wherein at least one non-natural nucleobase is isoguanine. 
     
     
         13 . The oligonucleotide primer according to  claim 1 , wherein at least one non-natural nucleobase is 5-methylisocytosine. 
     
     
         14 . The oligonucleotide primer according to  claim 1 , comprising at least four non-natural nucleobases. 
     
     
         15 . The oligonucleotide primer according to  claim 1 , wherein at least three of the four non-natural nucleobases are contiguous. 
     
     
         16 . The oligonucleotide primer according to  claim 1 , wherein at least one non-natural nucleobase is at the 3′ end of the tag. 
     
     
         17 . The oligonucleotide primer according to  claim 1 , wherein at least one non-natural nucleobase is not at the 5′ end of the tag. 
     
     
         18 . An oligonucleotide sequencing primer, comprising a 5′ non-target-specific universal sequencing tag comprising at least one non-natural nucleobase capable of hybridizing to a complementary non-natural nucleobase and capable of being replicated during amplification. 
     
     
         19 .- 32 . (canceled) 
     
     
         33 . An amplified polynucleotide sequencing template, comprising:
 (a) a polynucleotide sequence complementary to a region of a target polynucleotide;   (b) a 5′ universal sequencing tag comprising a plurality of non-natural nucleobases capable of hybridizing to a complementary non-natural nucleobase and capable of being replicated during amplification.   
     
     
         34 .- 48 . (canceled) 
     
     
         49 . A nucleic acid duplex, comprising an oligonucleotide primer comprising:
 (a) a 3′ target-specific nucleotide sequence;   (b) a 5′ non-target-specific universal tag comprising at least one non-natural nucleobase capable of complementary binding to a corresponding second non-natural nucleobase and capable of being replicated during amplification, wherein the oligonucleotide is hybridized to a polynucleotide target.   
     
     
         50 . A nucleic acid duplex, comprising:
 (a) a universal tagged primer comprising
 (i) a target-specific nucleotide sequence and 
 (ii) a 5′ universal tag comprising at least one non-natural nucleobase capable of being replicated during amplification and complementary to at least one corresponding non-natural nucleobase, 
   
       wherein the universal tagged primer is hybridized to
 (b) an amplified polynucleotide template comprising
 (i) a polynucleotide sequence complementary to a region of a target polynucleotide and 
 (ii) a 3′ universal tag comprising at least one non-natural nucleobase capable of being replicated during amplification. 
 
 
     
     
         51 .- 66 . (canceled) 
     
     
         67 . A method for replicating a polynucleotide target, comprising the step of hybridizing to a polynucleotide target an amplification primer comprising a 3′ target-specific nucleotide sequence and a 5′ universal sequencing tag comprising at least one non-natural nucleobase, wherein the non-natural nucleobase is capable of being replicated during amplification, and extending the amplification primer under PCR conditions, thereby producing a polynucleotide product that is complementary to the polynucleotide target and that further comprises a 5′ universal sequencing tag comprising at least one non-natural nucleobase which is capable of being replicated during amplification. 
     
     
         68 . A method according to  claim 67 , further comprising the step of sequencing the polynucleotide sequencing template using a universal sequencing primer complementary to the universal sequencing tag incorporated into the polynucleotide product, wherein the universal sequencing primer comprises a non-natural nucleobase complementary to a non-natural nucleobase of polynucleotide product. 
     
     
         69 . A method for sequencing a target polynucleotide, comprising:
 (a) providing an amplified polynucleotide sequencing template comprising
 (i) a 3′ universal sequencing tag comprising one or more non-natural nucleobase capable of being replicated during amplification, and 
 (ii) a 5′ target polynucleotide; and 
   (b) hybridizing the polynucleotide sequencing template with a universal sequencing primer at least a portion of [which is complementary to the 3′ universal sequencing tag of the polynucleotide sequencing template, wherein the universal sequencing primer comprises at least one non-natural nucleobase capable of being replicated during amplification and complementary to a non-natural nucleobase present in the 3′ universal sequencing tag,   (c) generating primer extension products under conditions sufficient to generate sequencing fragments corresponding to a portion of the polynucleotide target.   
     
     
         70 .- 85 . (canceled) 
     
     
         86 . A kit for sequencing a polynucleotide target, comprising:
 (a) an amplification primer comprising
 (i) a 3′ target-specific nucleotide sequence and 
 (ii) a 5′ non-target-specific universal sequencing tag comprising one or more non-natural nucleobases capable of complementary binding to a corresponding second non-natural nucleobase, wherein the one or more non-natural nucleobases and the corresponding second non-natural nucleobases are capable of being replicated during amplification; 
   (b) a universal sequencing primer comprising an oligonucleotide of the sequence comprising the 5′ non-target-sequencing tag.   
     
     
         87 .- 102 . (canceled)