Methods for sequencing polynucleotides
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.
1. A method of preparing a library for polynucleotide sequencing, comprising:
(a) providing a plurality of populations of target polynucleotide fragments, each of the target polynucleotide fragments having an internal nucleotide sequence and two ends, each of the target polynucleotide fragments being inserted into a circular vector; and
(b) cleaving the internal nucleotide sequence of each of the target polynucleotide fragments thereby removing the internal nucleotide sequence of each of the target polynucleotide fragments from the vector and leaving a segment of each of each end of the target polynucleotide fragments in the vector, thereby preparing providing a library for polynucleotide sequencing comprising a plurality of segments from the segment of each end of each of the target polynucleotide fragments in the vector after the cleaving step.
2. The method of claim 1 , further comprising determining the nucleotide sequences of at least one end of the segment of each of the target polynucleotide fragments in the vector after the cleaving step.
3. The method of claim 2 , wherein said determining the nucleotide sequences of at least one end of the segment of each of the target polynucleotide fragments in the vector comprises determining a nine base sequence located on the end of the segment.
4. The method of claim 2 , further comprising determining an ordering of the segments.
5. The method of claim 2 , wherein said determining the nucleotide sequences of at least one end of the segment of each end of the target polynucleotide fragments in the vector comprises performing a sequencing reaction on a DNA sequencer.
6. The method of claim 2 , further comprising concatenating the segments prior to said determining the nucleotide sequences of at least one end of the segment of each end of the target polynucleotide fragments in the vector.
7. The method of claim 1 , wherein the target polynucleotide fragments comprise genomic DNA sequences.
8. The method of claim 1 , wherein the target polynucleotide fragments are fragments resulting from a restriction endonuclease digest of a target polynucleotide.
9. The method of claim 1 , wherein step (b) further comprises circularizing the vector after said cleaving the internal nucleotide sequence of each of the target polynucleotide fragments.
10. The method of claim 1 , further comprising amplifying a segment from the library for polynucleotide sequencing by a polymerase chain reaction after the cleaving step.
11. The method of claim 1 , wherein the populations of target polynucleotide fragments are different.
12. The method of claim 1 , wherein the target polynucleotide fragments are different.
13. The method of claim 1 , wherein the circular vector comprises a first restriction endonuclease recognition site for a first restriction endonuclease flanking a 5′ end of the target polynucleotide fragments, and a second restriction endonuclease recognition site for a second restriction endonuclease flanking a 3′ end of the target polynucleotide fragments, and the cleavage sites of the first and second restriction endonucleases are within the internal nucleotide sequence of the target polynucleotide fragments, wherein the first and second restriction endonucleases are Type IIs restriction endonucleases.
14. The method of claim 13 , wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizes said first restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 8/12 between its restriction endonuclease recognition site and its cleavage site, and the second restriction endonuclease recognizes said second restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 8/12 between its recognition site and its cleavage site.
15. The method of claim 13 , wherein step (b) is performed with said first restriction endonuclease and said second restriction endonuclease.
16. The method of claim 13 , wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizes said first restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 12/13 between its restriction endonuclease recognition site and its cleavage site, and the second restriction endonuclease recognizes said second restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 12/13 reach between its recognition site and its cleavage site.
17. The method of claim 13 , wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizes said first restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 16/14 between its restriction endonuclease recognition site and its cleavage site, and the second restriction endonuclease recognizes said second restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 16/14 reach between its recognition site and its cleavage site.
18. The method of claim 13 , wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizes said first restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 20/18 between its restriction endonuclease recognition site and its cleavage site, and the second restriction endonuclease recognizes said second restriction endonuclease recognition site and cleaves the target polynucleotide fragments with a reach of 20/18 reach between its recognition site and its cleavage site.