US2005053972A1PendingUtilityA1

Method for identifying polymorphisms

57
Priority: Sep 5, 2000Filed: May 4, 2004Published: Mar 10, 2005
Est. expirySep 5, 2020(expired)· nominal 20-yr term from priority
Inventors:Vince P. Stanton, Jr.Jia Liu WolfeTomohiko KawateGregory L. VerdineJeffrey Olson
C07H 21/00C07H 19/14C12N 15/11C07H 19/10C12Q 1/6858C12P 19/34C12N 9/1252C12Q 1/683C07H 19/20C12Q 1/6827C12Q 1/6872
57
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Claims

Abstract

The present invention relates to methods for the detection of polymorphism in polynucleotides by using hybridization of fragments of segments of a polynucleotide suspected of containing a polymorphism with an oligonucleotide having a sequence complementary to a fragment identifying the polymorphism and subsequent detection of incorporated labels in the oligonucleotide-fragment duplex.

Claims

exact text as granted — not AI-modified
1 . A method for detecting polymorphism in a polynucleotide, comprising: 
 providing a polynucleotide suspected of containing a polymorphism;    amplifying a segment of the polynucleotide encompassing the suspected polymorphism wherein amplification comprises replacing one or more natural nucleotide(s), one of which is a nucleotide involved in the suspected polymorphism, at substantially each point of occurrence in the segment with a modified nucleotide or, if more than one natural nucleotide is replaced, with different modified nucleotides to form an amplified modified segment;    cleaving the amplified modified segment into fragments by contacting it with a reagent or reagents that cleave(s) the segment at substantially each point of occurrence of the modified nucleotide(s);    hybridizing the fragments to an oligonucleotide; and,    analyzing the hybridized fragments for an incorporated detectable label identifying the suspected polymorphism.    
     
     
         2 . The method of  claim 1 , wherein the detectable label is incorporated during amplification.  
     
     
         3 . The method of  claim 2 , wherein incorporating the detectable label during amplification comprises using a detectably labeled primer.  
     
     
         4 . The method of  claim 3 , wherein the detectably labeled primer comprises a radioactive primer or a primer containing a fluorophore.  
     
     
         5 . The method of  claim 1 , wherein incorporating the detectable label during amplification comprises using a detectably labeled, modified nucleotide.  
     
     
         6 . The method of  claim 5 , wherein the detectably labeled, modified nucleotide comprises a radioactive modified nucleotide or a modified nucleotide containing a fluorophore.  
     
     
         7 . The method of  claim 5 , wherein the detectably labeled, modified nucleotide is a detectably labeled, modified ribonucleotide.  
     
     
         8 . The method of  claim 7 , wherein the detectably labeled, modified ribonucleotide comprises a radioactive modified ribonucleotide or a modified ribonucleotide containing a fluorophore.  
     
     
         9 . The method of  claim 1 , wherein incorporating the detectable label during amplification comprises replacing a natural nucleotide, that is different than the natural nucleotide(s) being replaced with a modified nucleotide(s), at one or more point(s) of occurrence in the segment with a detectably labeled nucleotide.  
     
     
         10 . The method of  claim 9 , wherein the detectably labeled nucleotide comprises a radioactive nucleotide or a nucleotide containing a fluorophore.  
     
     
         11 . The method of  claim 9 , wherein the detectably labeled nucleotide comprises a detectably labeled ribonucleotide.  
     
     
         12 . The method of  claim 11 , wherein the detectably labeled ribonucleotide comprises a radioactive ribonucleotide or a ribonucleotide containing a fluorophore.  
     
     
         13 . The method of  claim 1 , wherein the detectable label is incorporated during cleavage.  
     
     
         14 . The method of  claim 13 , wherein incorporating the detectable label during cleavage comprises using detectably labeled tris(carboxyethyl)phosphine (TCEP).  
     
     
         15 . The method of  claim 14 , wherein using detectably labeled TCEP comprises using radioactive TCEP or TCEP containing a fluorophore.  
     
     
         16 . The method of  claim 13 , wherein incorporating the detectable label during cleavage comprises using a detectably labeled secondary amine.  
     
     
         17 . The method of  claim 16 , wherein using a detectably labeled secondary amine comprises using a radioactive secondary amine or a secondary amine containing a fluorophore.  
     
     
         18 . The method of  claim 1 , wherein the detectable label is incorporated during hybridization.  
     
     
         19 . The method of  claim 18 , wherein incorporating the detectable label during hybridization comprises hybridizing a second, detectably labeled oligonucleotide to the fragments hybridized to the oligonucleotide.  
     
     
         20 . The method of  claim 19 , wherein the second, detectably labeled oligonucleotide comprises a radioactive oligonucleotide or an oligonucleotide containing a fluorophore.  
     
     
         21 . The method of  claim 1 , wherein the detectable label is incorporated after cleavage or after hybridization, the method comprising: 
 cleaving using a reagent comprising TCEP or a secondary amine; and,    substituting the TCEP or secondary amine with a radioactive molecule or a fluorophore after cleavage or after hybridization.    
     
     
         22 . The method of  claim 1 , wherein the polymorphism is selected from the group consisting of a single nucleotide polymorphism (SNP), a deletion or an insertion.  
     
     
         23 . The method of  claim 1 , wherein amplifying the segment comprises a polymerase chain reaction (PCR).  
     
     
         24 . The method of  claim 1 , wherein amplifying the segment comprises replacing one natural nucleotide that is involved in the suspected polymorphism at each point of occurrence in the segment with a modified nucleotide to form a modified segment.  
     
     
         25 . The method of  claim 24 , wherein the modified nucleotide comprises a labeled, modified nucleotide.  
     
     
         26 . The method of  claim 25 , wherein the labeled modified nucleotide comprises a radioactive modified nucleotide or a modified nucleotide containing a fluorophore.  
     
     
         27 . The method of  claim 24 , wherein the modified nucleotide comprises a modified ribonucleotide.  
     
     
         28 . The method of  claim 24 , wherein the modified nucleotide comprises a labeled, modified ribonucleotide.  
     
     
         29 . The method of  claim 28 , wherein the labeled, modified ribonucleotide comprises a radioactive ribonucleotide or a ribonucleotide containing a fluorophore.  
     
     
         30 . The method of  claim 1 , wherein hybridizing the fragments to an oligonucleotide comprises using an oligonucleotide that is immobilized on a solid support.  
     
     
         31 . The method of  claim 1 , wherein the incorporated detectable label comprises fluorescence resonance energy transfer (FRET).

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