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 polynucleotide sequencing, comprising:
(a) providing a mixture of circular polynucleotides, wherein each of said circular polynucleotides comprises:
(i) a first Type IIs restriction endonuclease recognition site;
(ii) a target polynucleotide fragment; and
(iii) a second Type IIs restriction endonuclease recognition site,
wherein the first Type IIs restriction endonuclease recognition site is flanking a 5′ end of the target polynucleotide fragment, the second Type IIs restriction endonuclease recognition site is flanking a 3′ end of the target polynucleotide fragment, and the cleavage sites of the first and second Type IIs restriction endonucleases are within the interior of the target polynucleotide fragment,
wherein the first and second Type IIs restriction endonuclease recognition sites are arranged such that a first Type IIs restriction endonuclease recognizing said first Type IIs restriction endonuclease recognition site and a second Type IIs restriction endonuclease recognizing said second Type IIs restriction endonuclease recognition site cleave the target polynucleotide fragment, and
wherein said circular polynucleotides comprise different target polynucleotide fragments;
(b) cleaving the different target polynucleotide fragments contained in said mixture of circular polynucleotides with said first Type IIs restriction endonuclease, thereby producing restriction fragments comprising 5′ segments from the 5′ ends of the different target polynucleotide fragments;
(c) cleaving the different target polynucleotide fragments contained in said mixture of circular polynucleotides with said second Type IIs restriction endonuclease, thereby producing restriction fragments comprising 3′ segments from the 3′ ends of the different target polynucleotide fragments; and
(d) determining sequences of the 5′ segments and 3′ segments from the ends of the restriction fragments obtained from cleaving steps (b) and (c).
2. The method of claim 1 , wherein the first Type IIs restriction endonuclease recognition site and the second Type IIs restriction endonuclease recognition site are different.
3. The method of claim 1 , wherein the first Type IIs restriction endonuclease recognition site and the second Type IIs restriction endonuclease recognition site are the same.
4. The method of claim 1 , wherein each of the first Type IIs restriction endonuclease and the second Type IIs restriction endonuclease has a 16/14 reach between its recognition site and its cleavage site.
5. The method of claim 4 , wherein step (d) comprises determining a nine base sequence located on the end of each of the 5′ segments and the 3′ segments from the ends of the restriction fragments obtained from the cleaving steps (b) and (c).
6. The method of claim 1 , wherein the target polynucleotide fragments comprise genomic DNA sequences.
7. The method of claim 1 , wherein the target polynucleotide fragments comprise cDNAs.
8. The method of claim 7 , further comprising determining the frequency of expression of the cDNAs.
9. The method of claim 1 , further comprising determining an ordering of the 5′ segments and the 3′ segments from the ends of the restriction fragments obtained from the cleaving steps (b) and (c).
10. The method of claim 9 , further comprising constructing a physical map based on said ordering of the 5′ segments and the 3′ segments from the ends of the restriction fragments obtained from the cleaving steps (b) and (c).
11. The method of claim 1 , wherein said determining sequences of the 5′ segments and the 3′ segments from the ends of the restriction fragments obtained from the cleaving the steps (b) and (c) comprises performing a sequencing reaction on a DNA sequencer.
12. The method of claim 1 , wherein the target polynucleotide fragments are fragments resulting from a restriction endonuclease digest of a target polynucleotide.
13. The method of claim 1 , further comprising concatenating the 5′ segments and the 3′ segments from the ends of the restriction fragments obtained from the cleaving steps (b) and (c) prior to said determining sequences of the 5′ segments and the 3′ segments from the ends of the restriction fragments obtained from the cleaving steps (b) and (c).
14. The method of claim 1 , wherein the cleaving step (b) is prior to the cleaving step (c).
15. The method of claim 1 , wherein the cleaving step (b) is subsequent to the cleaving step (c).
16. The method of claim 1 , wherein the cleaving step (b) is concurrent with the cleaving step (c).
17. The method of claim 1 , wherein each of the first Type IIs restriction endonuclease and the second Type IIs restriction endonuclease has an 8/12 reach between its recognition site and its cleavage site.
18. The method of claim 1 , wherein each of the first Type IIs restriction endonuclease and the second Type IIs restriction endonuclease has a 12/13 reach between its recognition site and its cleavage site.
19. The method of claim 1 , wherein each of the first Type IIs restriction endonuclease and the second Type IIs restriction endonuclease has a 20/18 reach between its recognition site and its cleavage site.