Suppression of Non-Specific Amplification with High-Homology Oligonucleotides

Abstract

The invention comprises suppressor oligonucleotides for reducing amplification of a non-target nucleic acid sequences; the method of designing and using such oligonucleotides, as well as kits and reaction mixtures.

We claim: 
     
         1 . A suppressor oligonucleotide for use in a nucleic acid amplification reaction, having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism. 
     
     
         2 . The suppressor oligonucleotide of  claim 1 , wherein said at least one region of homology is at least 15 base pairs long. 
     
     
         3 . The suppressor oligonucleotide of  claim 1 , wherein said at least one region of homology spans the 3′-end of the suppressor oligonucleotide. 
     
     
         4 . The suppressor oligonucleotide of  claim 3 , wherein said at least one region of homology spanning the 3′-end of the suppressor oligonucleotide contains no more than 2 mismatches within 4 nucleotides from the 3′-end. 
     
     
         5 . The suppressor oligonucleotide of  claim 1 , selected from a group consisting of SEQ ID NOs: 1-5. 
     
     
         6 . A method of designing a suppressor oligonucleotide for use in a nucleic acid amplification reaction, comprising using sequence alignment algorithms to select an oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism. 
     
     
         7 . The method of  claim 6 , wherein said at least one region of homology is at least 15 base pairs long. 
     
     
         8 . The method of  claim 6 , wherein said at least one region of homology spans the 3′-end of the oligonucleotide. 
     
     
         9 . The method of  claim 8 , wherein said at least one region of homology spanning the 3′-end of the suppressor oligonucleotide contains no more than 2 mismatches within 4 nucleotides from the 3′-end. 
     
     
         10 . The method of  claim 6 , wherein the sequence alignment algorithm is selected from Basic Local Alignment Search Tool, Smith-Waterman process, ACANA, Bioconductor, FEAST, FASTA, REPuter and SWIFT BALSAM. 
     
     
         11 . The method of  claim 6 , further comprising:
 (a) identifying one or more regions of interest;   (b) for each region of interest, conducting a search of the target genome sequence using the region of interest as a query to identify regions of homology between the region of interest and the target genome;   (c) selecting sections of the region of interest having the most regions of homology in the target genome;   (d) designing one or more oligonucleotides in the sections selected in step (c);   (e) conducting a search of the target genome with the oligonucleotides designed in step (d) to identify the oligonucleotides with the maximum number of regions of homology to the target genome meeting one or both of the following criteria: at least 75% identity, no more than 2 mismatches are present within 4 nucleotides of 3′-terminal region of the oligonucleotide;   (f) optionally, conducting a search of the target genome with the oligonucleotides designed in step (d) to identify and exclude oligonucleotides having at least two regions of homology located on the opposite strands of the target genome, said regions of homology having at least 75% homology between the oligonucleotide and the target genome sequence, wherein said regions of homology are separated by fewer than approximately 1000 base pairs.   
     
     
         12 . A method of reducing amplification of a non-target nucleic acid template in a nucleic acid amplification reaction, comprising performing the amplification reaction in the presence of a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism. 
     
     
         13 . The method of  claim 12 , wherein said regions of homology are at least 15 base pairs long. 
     
     
         14 . The method of  claim 12 , wherein said regions of homology span the 3′-end of the oligonucleotide. 
     
     
         15 . The method of  claim 14 , wherein said regions of homology spanning the 3′-end of the suppressor oligonucleotide contain no more than 2 mismatches within 4 nucleotides from the 3′-end. 
     
     
         16 . The method of  claim 12 , wherein the suppressor oligonucleotide is selected from a group consisting of SEQ ID NOs: 1-5. 
     
     
         17 . A kit for performing an amplification reaction with reduced amplification, of the non-target sequences, comprising a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism. 
     
     
         18 . The kit of  claim 17 , further comprising one or more of the following: allele-specific primers, common primers, probes, nucleoside triphosphates, nucleic acid polymerase and buffers necessary for the function of the polymerase. 
     
     
         19 . The kit of  claim 17 , wherein the regions of homology have one or more of the following properties: at least 15 base pairs long; span the 3′-end of the suppressor oligonucleotide; within the last four base pairs of the 3′-end of oligonucleotide, the regions of homology contain no more than 2 mismatches. 
     
     
         20 . A reaction mixture for performing an amplification reaction with reduced amplification of the non-target sequences, comprising a suppressor oligonucleotide having a sequence comprising at least one region of homology with at least 75% identity to multiple sites in the genome of a target organism. 
     
     
         21 . The reaction mixture of  claim 20 , further comprising further comprising one or more of the following: allelic-specific primers, common primers, probes, nucleoside triphosphates, nucleic acid polymerase and buffers necessary for the function of the polymerase. 
     
     
         22 . The reaction mixture of  claim 20 , wherein the regions of homology have one or more of the following properties: at least 15 base pairs long; span the 3′-end of the suppressor oligonucleotide; within the last four base pairs of the 3′-end of the oligonucleotide, the regions of homology contain no more than 2 mismatches.