Method and compositions for ordering restriction fragments

• US 20050042673A1
• Filed: 10/08/2004
• Published: 02/24/2005
• Est. Priority Date: 02/23/1998
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Macevicz, Stephen C.
• Granted Patent: US 7,598,035 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 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

1-22. -22. (Cancelled)

23. A method of ordering pairs of sequence tags, the method comprising the steps of:

a) providing a population of pairs of sequence tags of restriction fragments, produced by digesting a fragment of genomic DNA with a plurality of combinations of restriction endonucleases;

b) removing duplicate pairs of sequence tags from the population;

c) selecting a pair of sequence tags from the population;

d) comparing each sequence tag of the selected pair with each sequence tag of a first pair and a last pair of a candidate ordering;

e) adding the selected pair to an end of the candidate ordering whenever a sequence tag of the selected pair matches the sequence tag of the first pair or the last pair of the candidate ordering, to form a new candidate ordering; and

f) repeating steps c) through e) until all pairs of the population have been selected.

24. The method of claim 23, wherein said population of pairs of sequence tags consists of n pluralities of pairs of sequence tags, each plurality being formed by digesting said fragment of genomic DNA in n separate reactions, each with a different n−

1 combination of restriction endonucleases, wherein each pair of sequence tags is formed by ligating a portion of each end of each restriction fragment together.

25. The method of claim 24, wherein said population of pairs of sequence tags consists of samples of pairs of sequence tags from each of said n pluralities.

26. The method of claim 25, wherein each of said samples has the same size.

27. The method of claim 26, wherein n=3 and each said restriction endonuclease has a six-basepair recognition site.

28-33. -33. (Cancelled)