Chemically-enhanced primer compositions, methods and kits

Abstract

A composition is provided comprising a negatively charged group, an oligonucleotide sequence and at least none or one nuclease-resistant linkage group to form a chemically-enhanced primer. The chemically-enhanced primer can be used for sequencing and fragment analysis. Methods for synthesizing the primer as well as a method of preparing DNA for sequencing and a method of sequencing DNA and kits containing the chemically-enhanced primer are also provided. The method of sequencing DNA can comprise contacting amplification reaction products with the composition under conditions in which excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded.

1 . A chemically-enhanced primer comprising: an oligonucleotide sequence having at least one nuclease-resistant linkage or no nuclease-resistant linkages and a negatively charged moiety (NCM) wherein the NCM is selected from a (Cn) spacer or a branched (Cn) spacer, wherein n can be any integer from 1 to 9. 
     
     
         2 . The chemically-enhanced primer of  claim 1 , wherein when the oligonucleotide comprises at least one nuclease-resistant linkage, the at least one nuclease-resistant linkage is at a terminal 3′ end of the oligonucleotide sequence. 
     
     
         3 . The chemically-enhanced primer of  claim 1 , wherein the NCM comprises a plurality of negatively charged moieties, and wherein the NCM is attached to a terminal 5′ end or within the oligonucleotide sequence. 
     
     
         4 .- 5 . (canceled) 
     
     
         6 . The chemically-enhanced primer of  claim 3 , wherein when n equals 3 the NCM comprises a (C3) x  spacer, wherein x equals at least 5, at least 6, at least 8, at least 9, at least 10, at least 15 at least 18 or more C3 spacers in a linear arrangement or [(C3) x ]z, wherein z equals 2 or 3 in a branched arrangement. 
     
     
         7 . The chemically-enhanced primer of  claim 6 , wherein the branched (C3) x  can be a doubler or a trebler. 
     
     
         8 . The chemically-enhanced primer of  claim 1 , wherein the oligonucleotide sequence comprises a universal primer sequence or a gene-specific primer sequence. 
     
     
         9 . The chemically-enhanced primer of  claim 8 , wherein the universal primer sequence is selected from M13, US1, T7, SP6, and T3. 
     
     
         10 . The chemically-enhanced primer of  claim 1 , wherein the chemically enhanced primer is resistant to digestion by a nuclease. 
     
     
         11 . The chemically-enhanced primer of  claim 1 , wherein the NCM can be a combination of [C3] x  and [C6] y  spacers, wherein y equals an integer from 1 to 20. 
     
     
         12 . The chemically-enhanced primer of  claim 1 , further comprising a dye-label. 
     
     
         13 . The chemically-enhanced primer of  claim 12 , wherein the dye-label is attached to the NCM or the oligonucleotide sequence. 
     
     
         14 . The chemically-enhanced primer of  claim 13 , wherein the dye-label is a fluorescent dye-label. 
     
     
         15 . The chemically-enhanced primer of  claim 10 , wherein the nuclease is selected from exonuclease I, Exo III, Pfu and DNA pol I. 
     
     
         16 - 35 . (canceled) 
     
     
         36 . A method of preparing DNA for sequencing, comprising the steps of:
 amplifying the DNA under conditions to produce amplification reaction products, the amplification reaction products comprising excess amplification primer;   contacting said amplification reaction products with a reaction mixture comprising a nuclease and a chemically-enhanced primer, whereby the excess amplification primer is degraded by the nuclease and the chemically-enhanced primer is essentially non-degraded, wherein the chemically-enhanced primer comprises at least one negative charged molecule (NCM) wherein the NCM is selected from a (Cn) spacer or a branched (Cn) spacer, wherein n can be any integer from 1 to 9, and an oligonucleotide sequence having at least one nuclease-resistant linkage or no nuclease-resistant linkages.   
     
     
         37 - 39 . (canceled) 
     
     
         40 . A method for sequencing DNA, comprising the steps of:
 a. amplifying DNA in a first reaction mixture comprising nuclease-sensitive amplification primers to form amplified DNA;   b. contacting the first reaction mixture of the amplifying step with a second reaction mixture comprising a nuclease and a chemically-enhanced primer, whereby the nuclease sensitive amplification primers are degraded by the nuclease;   c. inactivating the nuclease; and   d. reacting the amplified DNA in a sequencing reaction wherein the chemically-enhanced primer primes the sequencing reaction.   
     
     
         41 . The method of  claim 40 , further comprising:
 a. obtaining sequencing results based on the sequencing reaction; and   b. determining a nucleotide base sequence of the amplified DNA based on the results.   
     
     
         42 .- 43 . (canceled) 
     
     
         44 . The method of  claim 40 , wherein the chemically-enhanced primer comprises an oligonucleotide sequence having at least one nuclease-resistant linkage or no nuclease-resistant linkages, a negative charged molecule (NCM) selected from a (Cn) spacer or a branched (Cn) spacer, wherein n can be any integer from 1 to 9. 
     
     
         45 - 51 . (canceled) 
     
     
         52 . The method of  claim 40 , wherein the second reaction mixture further comprises a polymerase, deoxynucleotide triphosphates, dideoxynucleotide triphosphates and a dye-label. 
     
     
         53 .- 58 . (canceled) 
     
     
         53 . A kit comprising: a chemically-enhanced primer, comprising at least one negative charged molecule (NCM) wherein the NCM is selected from a (Cn) spacer or a branched (Cn) spacer, wherein n can be any integer from 1 to 9, and an oligonucleotide sequence having at least one nuclease-resistant linkage or no nuclease-resistant linkages. 
     
     
         60 .- 61 . (canceled) 
     
     
         62 . The chemically-enhanced primer of  claim 1 , having the structure of formula: 
       
         
           
           
               
               
           
         
       
     
     
         63 . The chemically-enhanced primer of  claim 1 , having the structure of one of the following formulae: