Methods for sequencing polynucleotides

• US 8,975,024 B2
• Filed: 10/02/2013
• Issued: 03/10/2015
• Est. Priority Date: 02/23/1998
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Macevicz, Stephen C.
• Primary Examiner(s): Lu, Frank
• Application Number:
  US14/044,413
• Publication Number:
  US 20140057274A1
• Time in Patent Office: 524 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/91.51, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.1
• 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 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;

(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 and leaving a segment of each end of the target polynucleotide fragments in the vector; and

(c) determining the nucleotide sequences of the segment of each end of the target polynucleotide fragments in the vector after the cleaving step.

2. The method of claim 1, wherein the target polynucleotide fragments comprise genomic DNA sequences.

3. The method of claim 1, wherein the target polynucleotide fragments are fragments resulting from a restriction endonuclease digest of a target polynucleotide.

4. 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.

5. The method of claim 1, further comprising amplifying a segment from an end of the target polynucleotide fragments by polymerase chain reaction after the cleaving step.

6. The method of claim 1, wherein the populations of target polynucleotide fragments are different.

7. The method of claim 1, wherein the target polynucleotide fragments are different.

8. 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.

9. The method of claim 8, wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizing said first restriction endonuclease recognition site 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 recognizing said second restriction endonuclease recognition site cleaves the target polynucleotide fragments with a reach of 8/12 between its recognition site and its cleavage site.

10. The method of claim 8, wherein step (b) is performed with said first restriction endonuclease and said second restriction endonuclease, thereby producing restriction fragments comprising segments from the ends of the target polynucleotide fragments.

11. The method of claim 8, wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizing said first restriction endonuclease recognition site 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 recognizing said second restriction endonuclease recognition site cleaves the target polynucleotide fragments with a reach of 12/13 reach between its recognition site and its cleavage site.

12. The method of claim 8, wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizing said first restriction endonuclease recognition site 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 recognizing said second restriction endonuclease recognition site cleaves the target polynucleotide fragments with a reach of 16/14 reach between its recognition site and its cleavage site.

13. The method of claim 8, wherein the first and second restriction endonuclease recognition sites are arranged such that the first restriction endonuclease recognizing said first restriction endonuclease recognition site 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 recognizing said second restriction endonuclease recognition site cleaves the target polynucleotide fragments with a reach of 20/18 between its recognition site and its cleavage site.

14. The method of claim 1, wherein step (c) comprises determining a nine base sequence located on the end of a segment from an end of the target polynucleotide fragments.

15. The method of claim 1, further comprising determining an ordering of the segments from the ends of the target polynucleotide fragments.

16. The method of claim 1, wherein said determining the nucleotide sequences of the segments of each end of the target polynucleotide fragments comprises performing a sequencing reaction on a DNA sequencer.

17. The method of claim 1, further comprising concatenating the segments from the ends of the target polynucleotide fragments prior to said determining the nucleotide sequences of the segments of each end of the target polynucleotide fragments in the vector.