Patent ID: 7169552

Claim:
A method for detecting a single nucleotide polymorphism located within an amplified AFLP target fragment, which is not an AFLP marker fragment that serves to distinguish between AFLP fingerprints, using an oligonucleotide sequence that is specific for said AFLP target fragment, and located adjacent (upstream) to the single nucleotide polymorphism to be detected, said method comprising the steps of: a) generating a mixture of PCR amplified restriction fragments using the AFLP method, by: digesting whole genomic DNA or cDNA with one or more restriction endonucleases to generate a mixture of fragments; ligating the restriction fragments obtained with at least one double stranded synthetic oligonucleotide adapter to generate tagged restriction fragments; and amplifying said tagged restriction fragments by PCR to generate a mixture of PCR amplified restriction fragments; b) contacting said mixture of amplified restriction fragments with said target-fragment specific oligonucleotide sequence under hybridization conditions, using a single target-fragment specific oligonucleotide per AFLP target fragment such that when the target restriction fragment is present, a hybrid is formed between the target restriction fragment and the oligonucleotide sequence, such that the resulting hybrid has at least one unpaired nucleotide of the target restriction fragment directly adjacent to the 3′ end of the oligonucleotide sequence, wherein said target-fragment specific oligonucleotide is complementary only to the intended target restriction fragment but not complementary to any other restriction fragment in the mixture; c) adding at least one labeled nucleotide or nucleotide analog to the mixture resulting from step b), under conditions suitable for extension of an oligonucleotide, such that when a hybrid of the target restriction fragment and the oligonucleotide is present, and said at least one labeled nucleotide or nucleotide analog is complementary to the at least one unpaired nucleotide of said target restriction fragment directly adjacent to the 3′ end of the oligonucleotide sequence, the nucleotide sequence is extended with the labeled nucleotide or nucleotide analog; and d) detecting the presence or absence of any hybrid(s) with (an) added labeled nucleotide(s) or nucleotide analog(s), or of any oligonucleotide sequence with (an) added labeled nucleotide(s) or nucleotide analog(s).