Amplification with primers of limited nucleotide composition

Abstract

The invention provides methods of amplification from a single primer or a pair of forward and reverse primers of limited nucleotide composition. Limited nucleotide composition means that the primers are underrepresented in at least one nucleotide type. Such primers have much reduced capacity to prime from each other or to extend initiated by mispriming from other than at their intended primer binding sites in a target nucleic acid.

1 . A method of amplifying a segment of a target nucleic acid comprising:
 contacting a sample comprising a target nucleic acid with forward and reverse primers; and   conducting an amplification reaction wherein an amplified segment of the target nucleic acid is formed by extension of the forward and reverse primers with the target nucleic acid serving as a template; wherein   the primers are underrepresented in one or more of the four standard nucleotide types, the underrepresented nucleotide type(s) being the same in the primers, and the amplified segment is the predominant amplification product formed from by extension of the forward and/or reverse primers.   
     
     
         2 . The method of  claim 1  wherein the target nucleic acid has a strand comprising a complement of a forward primer binding site and a reverse primer binding site. 
     
     
         3 . The method of  claim 1  wherein the target nucleic acid has a strand comprising a forward primer binding site and a reverse primer binding site. 
     
     
         4 . The method of  claim 1 , wherein the amplified segment constitutes at least 99% of all amplification products formed by extension of the forward and reverse primers. 
     
     
         5 . The method of  claim 1 , wherein the forward and reverse primers have greater complementarity to the forward and reverse primer bindings sites than to any other pair of primer binding sites supporting amplification in the sample. 
     
     
         6 . The method of  claim 1 , wherein the forward and reverse primers have one and only one of the four standard nucleotide types underrepresented. 
     
     
         7 . The method of  claim 1 , wherein the forward primer binding site and the reverse primer binding site are underrepresented in the complement of the underrepresented nucleotide type in the forward and reverse primers. 
     
     
         8 . The method of  claim 1 , wherein the forward and reverse primers have no more than two units of the underrepresented nucleotide type and the forward primer binding site and the reverse primer binding site have no more than four units of the complement of the underrepresented nucleotide type. 
     
     
         9 . The method of  claim 1 , wherein the forward and reverse primers have no more than one unit of the underrepresented nucleotide type and the forward primer binding site and the reverse primer binding site have no more than two units of the complement of the underrepresented nucleotide type. 
     
     
         10 . The method of  claim 1 , wherein the underrepresented nucleotide type(s) in the forward and reverse primers do not occupy the 3′ positions of the forward and reverse primers. 
     
     
         11 . The method of  claim 1 , wherein the forward and reverse primers consist of the three nucleotides other than the underrepresented nucleotide type and the forward primer binding site and the reverse primer binding site consist of the three nucleotides other than the complement of the underrepresent nucleotide in the forward and reverse primers. 
     
     
         12 . The method of any of  claim 1 , wherein the forward and reverse primer has one unit of underrepresented nucleotide at the 5′ end. 
     
     
         13 . The method of  claim 1 , wherein the forward and reverse primers have two of the standard four-nucleotide-types underrepresented. 
     
     
         14 . The method of  claim 13 , wherein the forward primer binding site and the reverse primer binding site are underrepresented in the complements of the underrepresented nucleotide types in the forward and reverse primers. 
     
     
         15 . The method of  claim 1 , wherein the complement of the forward primer binding site and the reverse primer binding site are contiguous. 
     
     
         16 . The method of  claim 1 , wherein the complement of the forward primer binding site and the reverse primer binding site are separated by a region excluding the underrepresented nucleotide type in the forward and reverse primers and its complement 
     
     
         17 . The method of  claim 1 , wherein the complement of the forward primer binding site and the reverse primer binding site are separated by a region including the underrepresented nucleotide in the forward and reverse primers or its complement or both. 
     
     
         18 . The method of  claim 1 , wherein the 3′ nucleotide of the forward and/or reverse primers is the complement of the underrepresented nucleotide in the forward and reverse primers. 
     
     
         19 . The method of  claim 1 , wherein the 3′ nucleotide of the forward and/or reverse primers is C or G. 
     
     
         20 . The method of  claim 1 , wherein the forward and/or reverse primer contains an unnatural nucleotide, which is inosine, isoC, isoG, 7-deaza-2′-deoxyguanosine, or 7-deaza-2′-deoxyadenosine. 
     
     
         21 - 84 . (canceled)