METHODS AND COMPOSITIONS FOR ORDERING RESTRICTION FRAGMENTS

• US 20100136548A1
• Filed: 09/11/2009
• Published: 06/03/2010
• Est. Priority Date: 02/23/1998
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Macevicz, Stephen C.
• Granted Patent: US 8,288,095 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

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.

Claims

1. 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.

2. The method of claim 1, wherein, wherein the first Type IIs restriction endonuclease recognition site and the second Type IIs restriction endonuclease recognition site have the same nucleotide sequence.

3. The method of claim 1, wherein determining the nucleotide sequence comprises performing a sequencing reaction on a DNA sequencer.

4. The method of claim 3, wherein determining the nucleotide sequence comprises performing a sequencing reaction on an APPLIED BIOSYSTEMS®

DNA sequencer.

5. The method of claim 1, wherein the ligated pair of Type IIs endonuclease cleavage sites comprises a ligated pair of different Type IIs endonuclease cleavage sites.

6. The method of claim 1, wherein the ligated pair of Type IIs endonuclease cleavage sites comprises a ligated pair of the same Type IIs endonuclease cleavage sites.

7. 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.

8. The mixture of claim 7, wherein the first Type IIs restriction endonuclease recognition site and the second Type IIs restriction endonuclease recognition site are different.

9. The mixture of claim 7, wherein the first Type IIs restriction endonuclease recognition site and the second Type IIs restriction endonuclease recognition site are the same.

10. The mixture of claim 7, wherein the mixture comprises 576 circular polynucleotides.

11. The mixture of claim 7, wherein the mixture comprises 4605 circular polynucleotides.

12. The mixture of claim 7, wherein the first Type IIs restriction endonuclease recognition site and the second Type IIs restriction endonuclease recognition site have a 16/14 reach.

13. The mixture of claim 7, wherein the polynucleotide insert comprises 1365 nucleotides.

14. The mixture of claim 7, wherein the polynucleotide insert comprises 2048 nucleotides.

15. The mixture of claim 7, wherein the polynucleotide insert comprises a genomic DNA sequence.

16. The mixture of claim 7, wherein the polynucleotide insert comprises a cDNA sequence.