Restriction enzyme genotyping

1. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, comprising:

(a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site;

(b) digesting the amplification product with the first restriction enzyme and the second restriction enzyme to generate a nucleic acid fragment having the selected polymorphic site within an overhanging end;

(c) treating the nucleic acid fragment generated in step (b) to convert the overhanging end to a blunt end by extension of the recessed portion of the overhanging end; and

(d) analyzing one or both strands of the nucleic acid fragment generated in step (c) to identify the nucleotide present at the selected polymorphic site.

2. A method for determining the nucleotide present at a selected polymorphic site in a target nucleic acid molecule, comprising:

(a) amplifying a portion of the target nucleic acid molecule comprising the selected polymorphic site using a first primer and a second primer, the second primer containing a recognition site for a first restriction enzyme and a recognition site for a second restriction enzyme, to generate amplification product containing a recognition site for the first restriction enzyme and a recognition site for the second restriction enzyme such that digestion of the amplification product with the first restriction enzyme and the second restriction enzyme generates a nucleic acid fragment containing the selected polymorphic site, wherein the cleavage site of the second restriction enzyme is such that the polymorphic site within the nucleic acid fragment is present within an overhanging end:

(b) digesting the amplification product with the second restriction enzyme in the presence of an enzyme that converts the overhanging end to a blunt end by extension of the recessed portion of the overhanging end under conditions which permit multiple cycles of digestion and conversion such that an oligonucleotide containing the polymorphic site is generated; and

(c) analyzing the oligonucleotide to identify the nucleotide present at the selected polymorphic site.

3. The method of claim 1 or claim 2 wherein the overhanging end is converted to a blunt end by the introduction of at least one mass modified nucleotide.

4. The method of claim 3 wherein the mass modified nucleotide is selected to base-pair with one of the possible nucleotides present at the polymorphic site within the overhanging end.

5. The method of claim 1 or claim 2 wherein the second primer comprises at least one nucleotide sequence that is not present in the target nucleic acid molecule.

6. The method of claim 5 wherein the second primer comprises 5′ nucleotide sequence that is complementary to a first portion of the target nucleic acid molecule, a 3′ nucleotide sequence that is complementary to a second portion of the target nucleic acid molecule, and a nucleotide sequence that is not complementary to the target nucleic acid molecule.

7. The method of claim 1 or claim 2 wherein either the first or the second restriction enzyme is a type IIS restriction enzyme.

8. The method of claim 1 or claim 2 wherein the step of analyzing the nucleic acid fragment to identify the nucleotide present at the selected polymorphic site comprises subjecting the nucleic acid fragment to mass spectrometry.

9. The method of claim 1 or 2 wherein the overhanging end is a 3′ end.

10. The method of claim 1 or 2 wherein the overhanging end is a 5′ end.

11. The method of claim 1 or 2 wherein the overhanging end is converted to a blunt end using a polymerase that fills the recessed end.

12. The method of claim 1 or 2 wherein the overhanging end is converted to a blunt end in the presence of ATP, CTP, GTP, and either TTP or UTP.