Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using AFLP®

Method for detecting a restriction fragment, comprising the steps of:

• (A) digesting a starting DNA, with one or more specific restriction endonucleases, to fragment said starting DNA into a series of restriction fragments;
• (B) ligating the restriction fragments thus obtained with one double-stranded oligonucleotide adapter to thereby produce tagged restriction fragments of the starting DNA;
• (C) contacting said tagged restriction fragments under hybridising conditions with at least one oligonucleotide primer;
• (D) amplifying said tagged restriction fragments hybridised with said primers by PCR so as to cause further elongation of the hybridised primers along the restriction fragments of the starting DNA to which said primers hybridised;
• and
• (E) identifying or recovering the amplified or elongated DNA fragment thus obtained;
• in which:
• the adapter used in step B) comprises within its sequence a recognition site for a IIS restriction endonuclease;
• and in which:
• step E) comprises the steps of:
• d) restricting the ligated dsDNA with the restriction endonuclease of the IIS type which recognizes the recognition site within the adapter so as to obtain at least a first and a second IIS-restricted dsDNA;
• e1) generating at least one ssDNA from at least one of the amplified restriction fragments obtained in step D);
• e2) detecting the at least one ssDNA generated in step e1).

Method for generating, and optionally detecting, an oligonucleotide that is specific for a starting nucleic acid, comprising the steps of:

• a) providing a dsDNA;
• b) restricting the dsDNA with at least one restriction endonuclease that restricts the dsDNA at two sites different from the recognition site of said restriction endonuclease, so as to provide a mixture of restricted fragments, said mixture comprising one or more fragments that contain the recognition site of the restriction endonuclease and one or more fragments that do not contain the recognition site of the restriction endonuclease used;
• c) ligating the first dsDNA to a second dsDNA, so as to provide a ligated dsDNA;
• d) amplifying the mixture of fragments obtained in step c);
• e) detecting at least one of the amplified fragments obtained in step d).

Method for generating, and optionally detecting, an oligonucleotide that is specific for a starting nucleic acid, comprising the steps of:

• a) providing a first dsDNA;
• b) restricting the first dsDNA with at least one restriction endonuclease that restricts the first dsDNA at two sites different from the recognition site of the restriction endonuclease, so as to provide a mixture of restricted fragments, the mixture comprising one or more fragments that contain the recognition site of the restriction endonuclease and one or more fragments that do not contain the recognition site of the restriction endonuclease;
• c) ligating the restricted fragments of the first dsDNA as obtained in (b) to a second dsDNA, in which the second dsDNA contains a recognition sequence for a type IIS restriction endonuclease in close proximity to the end ligating to the restricted fragment of the first dsDNA, so as to provide a ligated dsDNA;
• d) amplifying the ligated dsDNA obtained in (c);
• e) digestion of the amplified fragments obtained in (d) with the type IIS restriction endonuclease;
• f) detecting a fragment obtained in (e).

Method for generating, and optionally detecting, an oligonucleotide that is specific for a starting nucleic acid, comprising the steps of:

• a) providing a first dsDNA;
• b) restricting the first dsDNA with at least one restriction endonuclease that restricts the first dsDNA at two sites different from the recognition site of the restriction endonuclease, so as to provide a mixture of restricted fragments, the mixture comprising one or more fragments that contain the recognition site of the restriction endonuclease and one or more fragments that do not contain the recognition site of the restriction endonuclease;
• c) ligating ds DNA adapter sequences to both ends of the restriction fragments, so as to provide a ligated dsDNA;
• d) amplifying the ligated dsDNA obtained in (c);
• e) digestion of the amplified fragments obtained in (d) with the restriction endonuclease;
• f) detecting a fragment obtained in (e).