Methods and compositions for nucleic acid analysis

Abstract

The present invention relates to improved methods and composition for nucleic acid analysis. In particular, the present invention provides improved methods and compositions for carrying out nucleic acid analysis using modified nucleotides.

1 . A method for manufacturing oligonucleotide design data for use in selecting a region of a target nucleic acid to be amplified, wherein said region is selected to avoid or minimize the presence of natural nucleotides that can base-pair with a non-natural nucleotide, comprising: 
 a) providing a sequence of said target nucleic acid to a processor, wherein said processor is configured to analyze a sequence characteristic of said region selected from the group consisting of: location of said natural bases in said region, number of said natural bases in said region, and presence of said natural bases in said region; and    b) generating oligonucleotide design data using said analyzed sequence characteristic.    
   
   
       2 . The method of  claim 1 , wherein said natural base of said sequence characteristic is thymidine or uracil and wherein said non-natural nucleotide is iso-G.  
   
   
       3 . The method of  claim 2 , wherein said design data provides a primer sequence design so that an amplicon generated from said primer avoids iso-G/thymidine or uracil base pair matches within a region of the amplicon proximal to the primer.  
   
   
       4 . The method of  claim 2 , wherein said design data provides a region of the target nucleic acid avoiding iso-G/thymidine or uracil base pairs.  
   
   
       5 . The method of  claim 4 , wherein said region is at least 5 bases long.  
   
   
       6 . The method of  claim 4 , wherein said region is at least 10 bases long.  
   
   
       7 . The method of  claim 4 , wherein said region is at least 20 bases long.  
   
   
       8 . The method of  claim 4 , wherein said design data further provides probe or primer sequences for use in amplifying or detecting said region of said target nucleic acid.  
   
   
       9 . The method of  claim 1 , wherein said processor conducts calculations to assess the hybridization characteristics of a candidate probe, primer, or amplicon in the presence or absence of one or more iso-G/thymidine or uracil base pairs.  
   
   
       10 . A method for manufacturing a primer for use in a method of analyzing a target nucleic acid using non-natural nucleotides, wherein said method of analyzing a target comprises: 
 a) providing: 
 i) a first non-natural nucleotide;  
 ii) a sample suspected of comprising a target nucleic acid;  
 iii) a first amplification primer having a second non-natural nucleotide, said primer having a sequence selected to be upstream of a region of said target nucleic acid to be amplified, wherein said region is selected to avoid or minimize the presence of natural nucleotides that can base-pair with said first non-natural nucleotide;  
 iv) a second amplification primer; and  
 v) a polymerase;  
   b) exposing said sample to said first non-natural nucleotide, said first and second amplification primers, and said polymerase under conditions wherein an extension product is generated from at least said second amplification primer, wherein said extension product incorporates at least one of said first non-natural nucleotide,    said method for manufacturing a primer comprising selecting a primer sequence so as to amplify said region, wherein said region is selected to avoid or minimize the presence of natural nucleotides that can base-pair with said first non-natural nucleotide; and manufacturing a primer having said sequence.    
   
   
       11 . The method of  claim 10 , wherein said region is selected by the use of a processor configured to analyze a sequence characteristic of said region selected from the group consisting of: location of said natural bases in said region, number of said natural bases in said region, and presence of said natural bases in said region.  
   
   
       12 . The method of  claim 10 , wherein said natural nucleotide is thymidine or uracil and said non-natural nucleotide is iso-G.  
   
   
       13 . The method of  claim 10 , wherein said region is at least 5 nucleotides long.  
   
   
       14 . The method of  claim 10 , wherein said region is at least 10 nucleotides long.  
   
   
       15 . The method of  claim 10 , wherein said region is at least 20 nucleotides long.  
   
   
       16 . The method of  claim 10 , wherein said region is at least 50 nucleotides long.  
   
   
       17 . The method of  claim 10 , wherein said region lacks thymidine or uracil nucleotides.  
   
   
       18 . The method of  claim 10 , wherein said region has a low level of thymidine or uracil nucleotides.  
   
   
       19 . The method of  claim 18 , wherein said low level is less than 25%.  
   
   
       20 . The method of  claim 10 , wherein any thymidine or uracil nucleotides in said region are positioned so as to not have a detrimental impact on signal detection using said method of analyzing a target.