Oligonucleotide sizing using cleavable primers

Abstract

The present invention provides modified oligonucleotide primers designed to incorporate a cleavable moiety so that a 3′ portion of the primer (linked to an extension product) can be released from an upstream 5′ portion of the primer. Upon selective cleavage of the cleavable site, primer extension products that contain about five or fewer base pairs of the primer sequence are released, to provide more useful sizing and sequence information per fragment than extension products containing the entire primer.

1 . A method for determining presence of a polymorphism, comprising: 
 (a) hybridizing a primer, having a 5′ end and a 3′ end, with a target nucleic acid suspected of containing a polymorphism, wherein said primer has a first region containing the 5′ end of the primer and a second region containing the 3′ end of the primer and a cleavable site;    (b) extending the 3′ end of the primer with a polymerase in the presence of a nucleotide to generate an extension product;    (c) cleaving said extension product at the cleavable site to release an extension segment;    sizing the extension segment by mass spectrometry, whereby said cleaving is effective to increase the read length of the extension segment relative to the read length of the product of step (b); and    identifying any added nucleotides.    
     
     
         2 . The method of  claim 1 , wherein the first region of the primer further comprises an immobilization attachment site and the extension product is immobilized onto a solid support at the immobilization attachment site prior to the cleaving step.  
     
     
         3 . The method of  claim 2 , further comprising washing the extension product after said immobilizing and prior to said cleaving.  
     
     
         4 . The method of  claim 1 , wherein the nucleotide is selected from the group consisting of a deoxynucleotide, a chain-terminating nucleotide and a derivative thereof.  
     
     
         5 . The method of  claim 3 , wherein the nucleotide is a chain-terminating nucleotide.  
     
     
         6 . The method of  claim 5 , wherein the chain-terminating nucleotide is a dideoxynucleotide.  
     
     
         7 . The method of  claim 6 , wherein the dideoxynucleotide is selected from the group consisting of ddATP, ddTTP, ddUTP, ddGTP, ddITP and ddCTP.  
     
     
         8 . The method of  claim 5 , wherein the chain-terminating nucleotide is mass modified.  
     
     
         9 . The method of  claim 1 , wherein the target nucleic acid is DNA.  
     
     
         10 . The method of  claim 1 , wherein the target nucleic acid is RNA.  
     
     
         11 . The method of  claim 1 , wherein the target nucleic acid is immobilized.  
     
     
         12 . The method of  claim 11 , wherein the target nucleic acid is immobilized prior to said extending.  
     
     
         13 . The method of  claim 11 , wherein the target nucleic acid is immobilized after said extending.  
     
     
         14 . The method of  claim 1 , wherein the cleavable site is a blocking nucleotide capable of blocking 5′ to 3′ enzyme-promoted digestion, and where said cleaving is carried out by digesting the first region of the primer with an enzyme having a 5′ to 3′ exonuclease activity.  
     
     
         15 . The method of  claim 14 , wherein the enzyme is selected from the group consisting of T7 gene 6 exonuclease and phosphodiesterase.  
     
     
         16 . The method of  claim 14 , wherein the blocking nucleotide is selected from the group consisting of phosphorothioate, methyl phosphonate, phosphotriester, and peptide nucleic acid.  
     
     
         17 . The method of  claim 1 , where the cleavable site is selected from the group consisting of dialkoxysilane, 3′-(S)-phosphorothioate, 5′-(S)-phosphorothioate, 3′-(N)-hosphoramidate, 5′-(N)phosphoramidate, uracil, and ribose.  
     
     
         18 . The method of  claim 1 , wherein said sizing is by time-of-flight mass spectrometry.  
     
     
         19 . The method of  claim 1 , wherein a plurality of primers are hybridized to more than one target nucleic acid and the location of the cleavable site contained within the primers is varied to increase the mass difference between their respective extension segments.