DNA polymorphism identity determination using flow cytometry

Abstract

DNA polymorphism identity determination using flow cytometry. Primers designed to be immobilized on microspheres are allowed to anneal to the DNA strand under investigation, and are extended by either DNA polymerase using fluorescent dideoxynucleotides or ligated by DNA ligase to fluorescent reporter oligonucleotides. The fluorescence of either the dideoxynucleotide or the reporter oligonucleotide attached to the immobilized primer is measured by flow cytometry, thereby identifying the nucleotide polymorphism on the DNA strand.

What is claimed is:  
     
         1 . A method for determining the base identity at specific sites on a DNA strand, which comprises the steps of: 
 (a) synthesizing an oligonucleotide primer which can anneal to the DNA strand immediately adjacent to the base under investigation;    (b) immobilizing the oligonucleotide primer on microspheres;    (c) annealing the oligonucleotide primer to the DNA strand;    (d) incubating the microspheres to which the DNA strand has annealed to the immobilized oligonucleotide primer with fluorescent reporter molecules in the presence of an enzyme, each of the fluorescent reporter molecules having a labile base, whereby a fluorescent reporter molecule having a labile complementary base to the base under investigation covalently attaches to the immobilized oligonucleotide primer extending the immobilized oligonucleotide primer by one base unit; and    (e) analyzing the fluorescence of the microspheres using flow cytometry.    
     
     
         2 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 1 , wherein the fluorescent reporter molecules include fluorescent dideoxynucleotides and the enzyme includes DNA polymerase.  
     
     
         3 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 1 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with avidin.  
     
     
         4 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 1 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with streptavidin.  
     
     
         5 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 1 , wherein the oligonucleotide primers are covalently attached to the microspheres.  
     
     
         6 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 1 , wherein the oligonucleotide primers are hybridized to a complementary capture probe immobilized on microspheres.  
     
     
         7 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 1 , wherein the oligonucleotide primers include a sequence tag which is hybridized to a capture probe that is complementary thereto and is immobilized on the microspheres, and wherein the sequence tags and capture probes contain at least one of the non-natural bases iso-C and 5-methyl-iso-G.  
     
     
         8 . A method for determining the base identity at specific sites on a DNA strand, which comprises the steps of: 
 (a) synthesizing an oligonucleotide primer which can anneal to the DNA strand immediately adjacent to the base under investigation;    (b) immobilizing the oligonucleotide primer on microspheres;    (c) annealing the oligonucleotide primer to the DNA strand;    (d) incubating the microspheres to which the DNA strand has annealed to the immobilized oligonucleotide primer with fluorescent reporter molecules in the presence of an enzyme, each of the fluorescent reporter molecules having a labile base, whereby a fluorescent reporter molecule having a labile complementary nucleotide base to the base under investigation covalently attaches to the immobilized oligonucleotide primer;    (e) melting the DNA strand off of the ligated oligonucleotide; and    (f) analyzing the fluorescence of the microspheres using flow cytometry.    
     
     
         9 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 8 , wherein the reporter molecule includes fluorescent oligonucleotides and the enzyme includes DNA ligase.  
     
     
         10 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 8 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with avidin.  
     
     
         11 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 8 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with streptavidin.  
     
     
         12 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 8 , wherein the oligonucleotide primers are covalently attached to the microspheres.  
     
     
         13 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 8 , wherein the oligonucleotide primers are hybridized to a complementary capture probe immobilized on microspheres.  
     
     
         14 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 8 , wherein the oligonucleotide primers include a sequence tag which is hybridized to a capture probe that is complementary thereto and is immobilized on the microspheres, and wherein the sequence tags and capture probes contain at least one of the non-natural bases iso-C and 5-methyl-iso-G.  
     
     
         15 . A method for determining the base identity at specific sites on a DNA strand, which comprises the steps of: 
 (a) synthesizing an oligonucleotide primer which can anneal to the DNA strand immediately adjacent to the base under investigation;    (b) annealing the oligonucleotide primer to the DNA strand;    (c) incubating the annealed DNA strand and oligonucleotide primer with fluorescent reporter molecules in the presence of an enzyme, each of the fluorescent reporter molecules having a labile oligonucleotide base, whereby a fluorescent reporter molecule having a labile complementary base to the oligonucleotide base under investigation covalently attaches to the oligonucleotide primer extending the oligonucleotide primer by one base unit;    (d) immobilizing the extended oligonucleotide primers on microspheres;    and    (e) analyzing the fluorescence of the microspheres using flow cytometry.    
     
     
         16 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 15 , wherein the fluorescent reporter molecules include fluorescent dideoxynucleotides and the enzyme includes DNA polymerase.  
     
     
         17 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 15 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with avidin.  
     
     
         18 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 15 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with streptavidin.  
     
     
         18 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 15 , wherein the oligonucleotide primers are covalently attached to the microspheres.  
     
     
         19 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 15 , wherein the oligonucleotide primers are hybridized to a complementary capture probe immobilized on microspheres.  
     
     
         20 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 15 , wherein the oligonucleotide primers include a sequence tag which is hybridized to a capture probe that is complementary thereto and is immobilized on the microspheres, and wherein the sequence tags and capture probes contain at least one of the non-natural bases iso-C and 5-methyl-iso-G.  
     
     
         21 . A method for determining the base identity at specific sites on a DNA strand, which comprises the steps of: 
 (a) synthesizing an oligonucleotide primer can anneal to the DNA strand immediately adjacent to the base under investigation;    (b) annealing the oligonucleotide primer to the DNA strand;    (c) incubating the annealed DNA strand and oligonucleotide primer with fluorescent reporter molecules in the presence of an enzyme, each of the fluorescent reporter molecules having a labile base, whereby a fluorescent reporter molecule having a labile complementary nucleotide base to the base under investigation covalently attaches to the oligonucleotide primer;    (d) melting the DNA strand off of the ligated oligonucleotide primer;    (e) immobilizing the extended oligonucleotide primers on microspheres; and    (f) analyzing the fluorescence of the microspheres using flow cytometry.    
     
     
         22 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 21 , wherein the reporter molecules includes fluorescent oligonucleotides and the enzyme includes DNA ligase.  
     
     
         23 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 21 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with avidin.  
     
     
         24 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 21 , wherein the oligonucleotide primers are biotinylated and the microspheres are coated with streptavidin.  
     
     
         25 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 21 , wherein the oligonucleotide primers are covalently attached to the microspheres.  
     
     
         26 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 21 , wherein the oligonucleotide primers are hybridized to a complementary capture probe immobilized on microspheres.  
     
     
         27 . The method for determining the base identity at specific sites on a DNA strand as described in  claim 21 , wherein the oligonucleotide primers include a sequence tag which is hybridized to a capture probe that is complementary thereto and is immobilized on the microspheres, and wherein the sequence tags and capture probes contain at least one of the non-natural bases iso-C and 5-methyl-iso-G.