METHOD FOR THE DETECTION AND/OR ANALYSIS, BY MEANS OF PRIMER EXTENSION TECHNIQUES, OF SINGLE NUCLEOTIDE POLYMORPHISMS IN RESTRICTION FRAGMENTS, IN PARTICULAR IN AMPLIFIED RESTRICTION FRAGMENTS GENERATED USING AFLP3

摘要

The present invention relates to a method for determining the genotypes of polymorphic loci amplified in a mixture of restriction fragments, using an oligonucleotide sequence that is essentially complementary to part of the target restriction fragment, and located adjacent (upstream) to the polymorphism to be detected, said method comprising the steps of: a) contacting the mixture of restriction fragments with the oligonucleotide sequence under hybridization conditions, such that when the target restriction fragment is present, a hybrid is formed between the target restriction fragment and the oligonucleotide sequence, such that resulting hybrid has at least one unpaired nucleotide of the target restriction fragment directly adjacent to the 3' end of the oligonucleotide sequence; b) adding at least one labeled nucleotide or nucleotide analog to the mixture resulting from step a), 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; c) detecting the presence or absence of any hybrid(s) with (an) added labeled nucleotide(s) or nucleotide analog(s), and/or of any oligonucleotide sequence with (an) added labeled nucleotide(s) or nucleotide analog(s). The method of the invention can in particular be used for detecting single nucleotide polymorphisms (SNPs) in (constant) AFLP-fragments.