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 AFLP

摘要

The present invention relates to a method for determining the genotypes ofpolymorphic loci amplified in a mixture ofrestriction fragments, using an oligonucleotide sequence that is essentiallycomplementary to part of the target restriction fragment,and located adjacent (upstream) to the polymorphism to be detected, saidmethod comprising the steps of: a) contacting themixture of restriction fragments with the oligonucleotide sequence underhybridization conditions, such that when the target restrictionfragment is present, a hybrid is formed between the target restrictionfragment and the oligonucleotide sequence, such that resultinghybrid has at least one unpaired nucleotide of the target restriction fragmentdirectly adjacent to the 3' end of the oligonucleotidesequence; b) adding at least one labeled nucleotide or nucleotide analog tothe mixture resulting from step a), under conditionssuit-able for extension of an oligonucleotide; such that when a hybrid of thetarget restriction fragment and the oligonucleotide is present,and said at least one labeled nucleotide or nucleotide analog is complementaryto the at least one unpaired nucleotide of said targetrestriction fragment directly adjacent to the 3' end of the oligonucleotidesequence, the nucleotide sequence is extended with thelabeled nucleotide or nucleotide analog; c) detecting the presence or absenceof 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). Themethod of the invention can in particular be used for detecting singlenucleotide polymorphisms (SNPs) in (constant)AFLP-fragments.