Method and compositions for ordering restriction fragments

• US 7,598,035 B2
• Filed: 10/08/2004
• Issued: 10/06/2009
• Est. Priority Date: 02/23/1998
• Status: Expired due to Fees

• Current Assignee: Illumina Incorporated
• Original Assignee: Solexa Incorporated (Illumina Incorporated)
• Inventors: Macevicz, Stephen C.
• Primary Examiner(s): LU, FRANK WEI MIN
• Application Number:
  US10/962,337
• Publication Number:
  US 20050042673A1
• Time in Patent Office: 1,824 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/6.11
• 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 provides a method for 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 forming a linked pair of segments from each fragment of a population of polynucleotide fragments, wherein each said fragment is in a vector, said vector comprises two type IIs restriction endonuclease recognition sites, one of said type IIs restriction endonuclease recognition sites is adjacent to one end of the fragment and another of said type IIs restriction endonuclease recognition sites is adjacent to another end of the fragment, and one or more type IIs restriction endonucleases recognizing said type IIs restriction endonuclease recognition sites have two cleavage sites within the interior of the fragment, the method comprising:

treating each said vector and fragment with said one or more type IIs restriction endonucleases recognizing said type IIs restriction recognition sites, such that each fragment is cleaved at said cleavage sites, thereby removing the region of the fragment between said cleavage sites, whereby a pair of segments from said fragment remain connected to each other via said vector after said removing and each of said segments has a cleaved end; and

ligating together the cleaved ends produced by said removing thereby forming a linked pair of segments from each said fragment.

2. The method of claim 1 wherein the two type IIs recognition sites are recognized by the same type IIs restriction endonuclease.

3. A method of forming linearized vectors having pairs of segments from a population of polynucleotide fragments, the method comprising the steps of:

i) inserting each of said polynucleotide fragments from said population into a vector having two type IIs restriction endonuclease recognition sites and forming vectors containing said polynucleotide fragments such that each of the vectors containing said polynucleotide fragments comprises a fragment from said population of polynucleotide fragments, one of said type IIs restriction endonuclease recognition sites in each of the vectors containing said polynucleotide fragments is adjacent to one end of the fragment and another of said type IIs endonuclease recognition sites in each of the vectors containing said polynucleotide fragments is adjacent to another end of the fragment, and one or more type IIs restriction endonucleases recognizing said type IIs restriction endonuclease recognition sites have two cleavage sites within the interior of the fragment; and

ii) treating each of the vectors containing said polynucleotide fragments with said one or more type IIs restriction endonucleases recognizing the type IIs restriction endonuclease recognition sites and producing linearized vectors having pairs of segments wherein each of said linearized vectors having pairs of segments has a pair of segments of the fragment, one of said pair of segments is located at one end of each of the linearized vectors, and another of said pair of segments is located at another end of each of the linearized vectors.

4. The method of claim 3, wherein said type IIs recognition sites are recognized by the same type IIs restriction endonuclease.

5. The method of claim 3, further comprising the step of re-circularizing each of the linearized vectors, by ligating the pair of segments together.