Capturing sequences adjacent to type-IIS restriction sites for genomic library mapping
View Patent ↗The present invention relates to novel methods for sequencing and mapping genetic markers in polynucleotide sequences using Type-IIs restriction endonucleases. The methods herein described result in the “capturing” and determination of specific oligonucleotide sequences located adjacent to Type-IIs restriction sites. The resulting sequences are useful as effective markers for use in genetic mapping, screening and manipulation.
1. A method of determining a relative location of a target nucleotide sequence on a polynucleotide, the method comprising:
obtaining an ordered map of the polynucleotide according to a method comprising:
fragmenting the polynucleotide to generate a library of polynucleotide fragments;
identifying sequences in each of the polynucleotide fragments in the library, by a method comprising:
(a) cleaving the polynucleotide sequence with a first type-IIs endonuclease;
(b) ligating a first adapter sequence to the polynucleotide sequence cleaved in step (a), said first adapter having a recognition site for a second type-IIs endonuclease, the recognition site being oriented so that the second type-IIs endonuclease has a cleavage site in the polynucleotide sequence;
(c) cleaving the polynucleotide sequence resulting from step (b), with the second type-IIs endonuclease;
(d) ligating a second adapter sequence to the polynucleotide sequence cleaved in step (c); and determining the sequence of nucleotides of the polynucleotide sequence between the first and second adapter sequences;
comparing the sequences identified in each fragment with the sequences identified in each other fragment to obtain a level of correlation between each fragment and each other fragment; and
ordering the fragments according to their level of correlation to generate an ordered map of the polynucleotide;
(ii) fragmenting the polynucleotide;
(iii) determining which fragment generated in step (ii) includes the target nucleotide sequence; and
(iv) correlating a marker on the fragment identified in step (iii) with a marker on the ordered map to identify the approximate location of the target nucleotide sequence on the polynucleotide.
2. The method of claim 1 wherein:
the first type-IIs endonuclease is selected from the group consisting of BsmAI, EarI, MnlI, PleI, AlwI, BbsI, BsaI, BspMI, Esp3I, HgaI, SapI, SfaNI, BseRI, HphI and MboII; and
the second type-IIs endonuclease is selected from the group consisting of HgaI, BbvI, BspMI, BsmFI and FokI.
3. The method of claim 1 , wherein the sequence of nucleotides between the first and second adapter sequences in the fragments is determined by hybridization to an oligonucleotide probe, wherein said oligonucleotide probe is a positionally distinct probe on an oligonucleotide array, a position of the probe being indicative of the sequence of the probe.