Polymerase extension at 3' terminus of PNA-DNA chimera

Abstract

The invention provides methods and kits for primer extension of PNA-DNA chimera from template nucleic acids using polymerases, nucleotide 5′-triphosphates, and primer extension reagents. Structural requirements of the chimera for primer extension include 5 to 15 contiguous PNA monomer units, 3 or more contiguous nucleotides, and a 3′ hydroxyl terminus. The chimera and/or a nucleotide is labelled with fluorescent dyes or other labels. The methods include DNA sequencing, DNA fragment analysis, reverse transcription, mini-sequencing, chromosome labelling, amplification, and single nucleotide polymorphism (SNP) detection.

1 . A labeled polymerase extended PNA-DNA chimera comprising the structure:  
         P x -L-N y  3′ Extension  
       wherein: 
 P is independently a PNA monomer;  
 X is an integer from 5 to 15;  
 L represents a covalent linkage between P and N;  
 each N is independently a nucleotide;  
 y is an integer from 1-15; and  
 3′ Extension is the polymerase extension product comprising at least one non-radioisotopically labeled nucleotide wherein the label is selected from the group consisting of a fluorescent dye, a fluorescence quencher, a hybridization stabilizer, an energy-transfer dye pair, an electrophoretic mobility modifier, a chemiluminescent dye, an amino acid, a protein, a peptide, an enzyme and an affinity ligand.  
 
     
     
         2 . The PNA-DNA chimera of  claim 1 , wherein the PNA-DNA chimera is hybridized to the template nucleic acid.  
     
     
         3 . The PNA-DNA chimera of  claim 2 , wherein the template comprises an affinity capture ligand.  
     
     
         4 . The PNA-DNA chimera of  claim 1 , wherein the label is a fluorescent dye.  
     
     
         5 . The PNA-DNA chimera of  claim 4 , wherein the moiety represented by P x -L-N y  is unlabeled.  
     
     
         6 . The PNA-DNA chimera of  claim 1 , wherein the moiety represented by P x -L-N y  is unlabeled.