High throughput detection of molecular markers based on aflp and high throughput sequencing

摘要

Method for the identifiying the presence or absence of restriction fragments in a sample, comprising the steps of (a) providing two or more sample nucleic acids; (b) digesting each sample nucleic acid with at least one restriction endonuclease to obtain a set of restriction fragments; (c) providing double stranded synthetic adaptors comprising - a 5" primer- complementary sequence, a sample-specitic identifier section, - at least one end that which can be ligated to the blunt or protruding end of a restriction fragment; (d) ligating the double stranded synthetic adaptors to the restriction fragments in the set, to provide a set of adaptor- ligated restriction fragments; (e) optionally, amplification of the set of adaptor-ligated restriction fragments, with one or more primers that are at least complementary to: - the 5" primer-complementary sequence of the adaptor, - the sample-specific identifier section of the adaptor, and, - optionally, a section of the adaptor that is complementary to the protruding remains of the recognition sequence of the at least one restriction endonuclease, to provide for amplified adaptor-ligated restriction fragments (amplicons) ; (f) determining the sequence of at least the sample-specific identifier section, the remains of the recognition sequence of the at least one restriction endonuclease and part of the sequence of the restriction fragment located adjacent to the adaptor derived sequence or adjacent to the remains of the recognition sequence of the at least one restriction endonuclease, (g) comparing two or more samples for the presence or absence of adapter-ligated restriction fragments; (h) identifying the presence or absence of adaptor-ligated restriction fragments in the sample.