Methods and compositions for ordering restriction fragments
AssigneeIlluminia, Inc.
InventorStephen C. Macevicz
Life of this patent
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Abstract
The invention relates to methods and systems for sequencing and constructing a high resolution physical map of a polynucleotide. In accordance with the invention, nucleotide sequences are determined at the ends of restriction fragments produced by a plurality of digestions with a plurality of combinations of restriction endonucleases so that a pair of nucleotide sequences is obtained for each restriction fragment. A physical map of the polynucleotide is constructed by ordering the pairs of sequences by matching the identical sequences among the pairs.
Claims16
A method of sequencing a polynucleotide insert, comprising:
- providing a circular polynucleotide comprising a polynucleotide insert having a 5′ end and a 3′ end, wherein a first Type IIs restriction endonuclease recognition site is adjacent the 3′ end and a second Type IIs restriction endonuclease recognition site is adjacent the 5′ end, and wherein the insert comprises a ligated pair of Type IIs endonuclease cleavage sites formed from cleavage of the insert with one or more Type IIs endonucleases, followed by ligation of the cleavage sites:
- and
- determining the nucleotide sequence of the polynucleotide insert.
A mixture of different circular polynucleotides, each of the polynucleotides comprising:
- a first Type IIs restriction endonuclease recognition site;
- a polynucleotide insert that differs from other circular polynucleotides in the mixture;
- and
- a second Type IIs restriction endonuclease recognition site,
- wherein the first Type IIs restriction endonuclease recognition site is adjacent to a first end of the polynucleotide insert and the second Type IIs restriction endonuclease recognition site is adjacent to a second end of the polynucleotide insert, and
- wherein each Type IIs restriction endonuclease recognition site is oriented such that a Type IIs restriction endonuclease recognizing either site cleaves the polynucleotide insert.
Dependencies are inferred from the claim text.