Method for detecting single nucleotide polymorphisms (SNPs) and point mutations

Abstract

A method of genotyping single nucleotide polymorphisms (SNP) and point mutations in nucleic acid based on chain extension by polymerase. This invention is based on the fact that the nucleoside immediately 5′ adjacent to any SNP/point mutation site is known, and the neighboring sequence immediately 3′ adjacent to the site is also known. A primer complimentary to the sequence directly adjacent the 3′ side of the SNP in a target polynucleotide is used for chain elongation. The polymerase reaction mixture contains one chain terminating nucleotide having the base complimentary to nucleotide directly adjacent to the 5′ side of the SNP of the target polynucleotide. An additional dNTP may be added to produce a primer with the maximum of two base extension. The resultant elongation/termination reaction product are analysed for incorporation of the chain terminator nucleotide or for chain length extension of the primer.

1 ) a method of analysing the base identity of a target nucleotide in a target polynucleotide molecule, said target polynucleotide molecule having a 3′ portion, a 5′ portion and said target nucleotide therebetween, said method comprising: 
 (a) reacting said target polynucleotide molecule with a primer oligonucleotide, said primer oligonucleotide having a sequence complimentary to a section of said 3′ portion directly adjacent to said target nucleotide;  
 (b) providing a chain termination nucleotide having a base that is complimentary to the base of the nucleotide directly adjacent to the 5′ side of said target nucleotide;  
 (c) elongating said primer across said target nucleotide under appropriate polymerisation condition using a chain extending agent, said chain terminating nucleotide having functional residues to allow said extending agent to incorporate said chain terminating nucleotide onto the 3′ end of said primer, said chain terminating nucleotide further terminating said elongation reaction by blocking further extension of said primer after said chain terminating nucleotide is incorporated;  
 (d) analysing said elongation/termination reaction for primer oligonucleotides containing incorporated chain terminator nucleotides.  
 
     
     
         2 ) A method according to claim ( 1 ), wherein step (d) involves analysing said elongation/termination reaction for oligonucleotides that have zero, one or two bases longer than said primer.  
     
     
         3 ) A method according to claim ( 2 ) wherein said chain terminating nucleotide is a 2′,3′-dideoxyribonucleoside 5′-triphosphate and said extending agent is DNA polymerase.  
     
     
         4 ) A method according to claim ( 2 ) wherein said target polynucleotide molecule is amplified nucleic acid.  
     
     
         5 ) A method according to claim ( 1 ) wherein a second reaction mixture is provided, said second reaction mixture containing all the reagents as those mentioned in claim ( 1 ), said second reaction mixture further containing an extender nucleotide with a second base having an identity different from said base of said chain terminating nucleotide, said extender nucleotide capable of supporting further chain elongation after incorporation into said oligonucleotide, said method further including the steps of elongating said primer oligonucleotide in said second reaction mixture using said extending agent under appropriate reaction conditions, and analysing said second reaction mixture for primer oligonucleotides containing incorporated chain termination nucleotide.  
     
     
         6 ) A method according to claim ( 1 ) wherein one, two or three additional reaction mixtures are provided, each said additional reaction mixture containing all the reagents as those mentioned in claim ( 1 ), each said additional reaction mixture further containing an extender nucleotide with a unique base having an identity different from said base of said chain terminating nucleotide, each said extender nucleotide capable of supporting further chain elongation after incorporation into said primer oligonucleotide, said method further including the step of elongating said primer oligonucleotide in said additional mixture using said extending agent under suitable reaction conditions, and analysing said additional reaction mixture for oligonucleotides containing incorporated chain terminator nucleotide.  
     
     
         7 ) A method according to claim ( 5 ) wherein said primer oligonucleotides are analysed for zero, one or two bases extension after said elongation/termination reaction.  
     
     
         8 ) A method according to claim ( 6 ) wherein said primer oligonucleotides are analysed for zero, one or two bases extension after said elongation/termination reaction.  
     
     
         9 ) A method of detecting single nucleotide polymorphism comprising: 
 (a) obtaining double-strand nucleic acid from a source for which said single nucleotide polymorphism analysis method is to be performed;    (b) amplifying a portion of said nucleic acid containing said single nucleotide polymorphism by polymerase chain reaction using a first primer and a second primer;    (c) separating said first and second primers from said amplification product;    (d) mixing said amplification product with a reaction mixture, said amplification product having a 3′ portion, a 5′ portion and a target nucleotide therebetween, said target nucleotide having said single nucleotide polymorphism;    (e) providing in said reaction mixture a third primer oligonucleotide molecule having a sequence complimentary to a section of said 3′ portion directly adjacent to said target nucleotide;    (f) providing in said reaction mixture a polymerase enzyme capable of extending the 3′ end of said third primer;    (g) providing in said reaction mixture a chain terminating nucleotide, said chain terminating nucleotide having functional residues to allow said polymerase to polymerise said chain terminating nucleotide onto the 3′ end of that primer while blocking further extension of said primer after said chain terminating nucleotide is incorporated; said chain terminating nucleotide further having a base that is complimentary to the base of the nucleotide directly adjacent the 5′ side of said target nucleotide;    (h) reacting said polymerase with said third primer and said amplification product in said reaction mixture under appropriate reaction conditions;    (i) analyzing said reaction mixture for oligonucleotides that have zero, one or two bases longer than said third primer.    
     
     
         10 ) A method according to claim ( 9 ) wherein a second reaction mixture is provided, said second reaction mixture containing all the reagents in said second reaction mixture, said second reaction mixture further containing an extender nucleotide containing a second base having an identity different from said base of said chain terminating nucleotide, said extender nucleotide capable of supporting further chain elongation after incorporation into said third primer by said polymerase, said method further including the steps of reacting said polymerase in said second reaction mixture, and analysing said second reaction mixture for oligonucleotides that have zero, one or two bases longer than said primer.  
     
     
         11 ) An oligonucleotide for detecting single nucleotide polymorphism located at a target site on a target polynucleotide, said oligonucleotide having a sequence complimentary to a sequence directly adjacent to the 3′ side of said target site.  
     
     
         12 ) An oligonucleotide according to claim ( 12 ) wherein the length of said oligonucleotide is between 15-55 bases.  
     
     
         13 ) The method according to claim ( 1 ) wherein the 5′ end of said primer oligonucleotide is attached to a solid surface.  
     
     
         14 ) A method according to claim ( 9 ) wherein said third primer oligonucleotide molecule is attached to a solid surface.