Method and compositions for ordering restriction fragments

• US 6,720,179 B1
• Filed: 04/14/2000
• Issued: 04/13/2004
• Est. Priority Date: 02/23/1998
• Status: Expired due to Term

• Current Assignee: Illumina Incorporated
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Macevicz, Stephen C.
• Primary Examiner(s): Whisenant, Ethan
• Assistant Examiner(s): Lu, Frank
• Application Number:
  US09/549,748
• Time in Patent Office: 1,460 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/320.1, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/320.1
• 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 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 vector containing a restriction fragment of DNA, the vector having a top strand and a bottom strand, and comprising, in sequence:

a first type IIs recognition site oriented in the 5′

to 3′

direction of said top strand,

said restriction fragment, and

a second type IIs recognition site oriented in the 5′

to 3′

direction of said bottom strand,

such that a first type IIs restriction endonuclease recognizing the first type IIs recognition site has a cleavage site which is 3′

of the first recognition site in the top strand 5′

to 3′

direction, and is within the restriction fragment,

and a second type IIs restriction endonuclease recognizing the second type IIs recognition site has a cleavage site which is 3′

of the second recognition site in the bottom strand 5′

to 3′

direction, and is within the restriction fragment.

2. The vector of claim 1, further including a third restriction site and a fourth restriction site, for accepting said fragment of DNA, the third restriction site being between said first type IIs recognition site and said cleavage site of said first type IIs recognition site, and the fourth restriction site being between said second type IIs recognition site and said cleavage site of said second type IIs recognition site.

3. The vector of claim 2, further including a third type IIs recognition site, a third type IIs cleavage site, a fourth type IIs recognition site and a fourth type IIs cleavage site, wherein the third type IIs cleavage site is identical to that of the third restriction site of claim 2, and the fourth type IIs cleavage site is identical to that of the fourth restriction site of claim 2.

4. The vector of claim 1, wherein said first type IIs restriction endonuclease and second type IIs restriction endonuclease are the same.

5. The vector of claim 3, wherein said first and second type IIs recognition sites are BsgI sites, said third and fourth restriction sites are HindIII and EcoRI sites, respectively, and said third and fourth type IIs recognition sites are BbvI sites, as shown in the following sequence (SEQ ID NO:

6);