Nucleic acid constructs and methods of use

Abstract

The present invention provides oligonucleotide constructs, sets of such oligonucleotide constructs, and methods of using such oligonucleotide constructs to provide validated sequences or sets of validated sequences corresponding to desired ROIs. Such validated ROIs and constructs containing these have a wide variety of uses, including in synthetic biology, quantitative nucleic acid analysis, polymorphism and/or mutation screening, and the like.

1 - 29 . (canceled) 
     
     
         30 . An amplification reaction mixture, comprising:
 a plurality of nucleic acids each comprising a region of interest (ROI) and a primer binding site; and   a plurality of primers, wherein each primer: i) hybridizes to the primer binding site of a nucleic acid of the plurality of nucleic acids, and ii) comprises a unique identifier,   wherein the plurality of primers and the number of different unique identifiers therein are in excess relative to the number of nucleic acids in the plurality of nucleic acids, such that a particular unique identifier has a high probability of being unique in its association with a particular ROI in an amplification product of the amplification reaction mixture.   
     
     
         31 . The amplification reaction mixture of  claim 30 , wherein the unique identifier in the amplification product distinguishes the amplification product from other amplification products of the amplification reaction mixture comprising substantially the same ROI. 
     
     
         32 . The amplification reaction mixture of  claim 30 , wherein at least two of the amplification products of the amplification reaction mixture comprise substantially the same ROI and different unique identifiers. 
     
     
         33 . The amplification reaction mixture of  claim 30 , wherein at least five of the amplification products of the amplification reaction mixture comprise substantially the same ROI and different unique identifiers. 
     
     
         34 . The amplification reaction mixture of  claim 30 , wherein each of the unique identifiers comprises a degenerate sequence. 
     
     
         35 . The amplification reaction mixture of  claim 34 , wherein the number of nucleotides in the degenerate sequence is such that the number of potential and actual sequences in the unique identifier is greater than the number of nucleic acids in the plurality of nucleic acids. 
     
     
         36 . The amplification reaction mixture of  claim 30 , wherein the unique identifiers are selected from a diverse, pre-defined set of sequences. 
     
     
         37 . The amplification reaction mixture of  claim 30 , wherein the amplification product comprises at least one amplification site. 
     
     
         38 . The amplification reaction mixture of  claim 30 , wherein the amplification product comprises two or more amplification sites flanking the ROI and the unique identifier. 
     
     
         39 . The amplification reaction mixture of  claim 38 , wherein the two or more amplification sites comprise a binding site for a universal primer and a binding site for a subset-specific primer. 
     
     
         40 . The amplification reaction mixture of  claim 30 , wherein the amplification product comprises a single amplification site adjacent to the ROI and the unique identifier. 
     
     
         41 . The amplification reaction mixture of  claim 30 , wherein the amplification product comprises one or more excision sites. 
     
     
         42 . The amplification reaction mixture of  claim 30 , wherein the amplification product comprises at least two excision sites flanking the ROI. 
     
     
         43 . The amplification reaction mixture of  claim 30 , wherein one or more of the nucleic acid constructs comprises a single excision site adjacent to the ROI. 
     
     
         44 . The amplification reaction mixture of  claim 30 , wherein the plurality of nucleic acids are in solution. 
     
     
         45 . The amplification reaction mixture of  claim 30 , wherein the plurality of nucleic acids are immobilized on a support. 
     
     
         46 . The amplification reaction mixture of  claim 30 , wherein the plurality of nucleic acids are isolated from a biological sample and comprise a rare sequence variant. 
     
     
         47 . The amplification reaction mixture of  claim 46 , wherein the rare sequence variant is a low-frequency mutation or a minor allelic variant or haplotype. 
     
     
         48 . The amplification reaction mixture of  claim 30 , wherein the plurality of primers comprise primers for a two-primer polymerase chain reaction (PCR). 
     
     
         49 . The amplification reaction mixture of  claim 30 , wherein the plurality of primers comprise primers for an asymmetric polymerase chain reaction (PCR).