Method and compositions for ordering restriction fragments

• US 20040082001A1
• Filed: 11/12/2003
• Published: 04/29/2004
• Est. Priority Date: 02/23/1998
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Macevicz, Stephen C.
• Application Number:
  US10/706,118
• Publication Number:
  US 20040082001A1
• 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. A method of mapping a polynucleotide, the method comprising the steps of:

(a) providing a plurality of populations of restriction fragments, the restriction fragments of each population having ends defined by digesting the polynucleotide with a plurality of combinations of restriction endonucleases;

(b) determining the nucleotide sequence of a portion of each end of each restriction fragment of each population so that a pair of nucleotide sequences is obtained for each restriction fragment of each population; and

(c) ordering the pairs of nucleotide sequences by matching the nucleotide sequences between pairs to form a map of the polynucleotide.

2. The method of claim 1 wherein said step of determining said nucleotide sequence of said end of each restriction fragment includes the steps of enzymatically removing a segment of nucleotides from each said end;

ligating the segment of nucleotides from each said end together to form a pair of segments, ligating a sample of pairs of segments from said plurality of populations to form one or more concatenations of pairs of segments, and sequencing the concatenations of pairs of segments.

3. The method of claim 2 wherein said plurality of populations of restriction fragments is between 3 and 8, inclusive.

4. The method of claim 3 wherein said combinations of restriction endonucleases used for digesting said polynucleotide consist of three restriction endonucleases selected from a set of four restriction endonucleases.

5. The method of claim 3 wherein said combinations of restriction endonucleases used for digesting said polynucleotide consist of two restriction endonucleases selected from a set of three restriction endonucleases.

6. The method of claim 5 wherein said steps of enzymatically removing said segments of nucleotides is carried out with one of more type IIs restriction endonucleases.

7. The method of claim 6 wherein said segments of nucleotides consist of between 7 and 9 nucleotides.

8. A physical map of a polynucleotide comprising an ordered series of segments of nucleotides, the segments of nucleotides being adjacent to and including recognition sites in the polynucleotide of a plurality of predetermined restriction endonucleases.

9. The physical map of claim 8 wherein said ordered series comprises at least one hundred said segments of nucleotides and wherein said plurality of predetermined restriction endonucleases is between 2 and 6, inclusive.

10. The physical map of claim 9 wherein said segments of nucleotides of said ordered series are separated by unsequenced regions of said restriction fragments.

11. The physical map of claim 10 wherein said ordered series comprises at least one thousand said segments of nucleotides.

12. The physical map of claim 8 wherein said polynucleotide is between 180 kilobases and 1 megabase in length.

13. The physical map of claim 8 wherein said polynucleotide is a genome of a microorganism.

14. A method of mapping a polynucleotide, the method comprising the steps of:

(a) providing a plurality of populations of restriction fragments, the restriction fragments of each population having an interior and ends defined by digesting the polynucleotide with a plurality of combinations of restriction endonucleases, and each restriction fragment being inserted into a vector;

(b) cleaving each vector to remove the interior of the restriction fragment and to leave a segment of each end of the restriction fragment in the vector;

(c) circularizing each vector so that the segments of each end of each restriction fragment are ligated together to form a pair of segments;

(d) determining the nucleotide sequences of a sample of pairs of segments to obtain a sample of pairs of nucleotide sequences; and

(e) ordering the pairs of nucleotide sequences by matching the nucleotide sequences between pairs to form a map of the polynucleotide.

15. The method of claim 14 wherein said step of determining said nucleotide sequences of said sample of said pairs of segments includes the steps of ligating said sample of pairs of segments from said plurality of populations to form one or more concatenations of pairs of segments, and sequencing the concatenations of pairs of segments.

16. The method of claim 15 wherein said sample includes a number of said pairs of segments large enough so that with a probability of ninety-nine percent every possible kind of pair of segments is represented in said sample.

17. The method of claim 16 wherein said step of cleaving is carried out with one or more type IIs restriction endonucleases.

18. A vector for cloning a fragment of DNA, the vector comprising:

a first type IIs restriction site and a second type IIs restriction site such that a first type IIs restriction endonuclease recognizing the first type IIs restriction site has a cleavage site downstream from the first type IIs restriction site and such that a second type IIs restriction endonucleases recognizing the second type IIs restriction site has a cleavage site upstream from the second type IIs site.

19. The vector of claim 18 further including a third restriction site and a fourth restriction site for accepting said fragment of DNA, the third restriction site being immediately downstream of said first type IIs restriction site such that said cleavage site of said first type IIs restriction endonuclease is downstream of the third restriction site, and the fourth restriction site being immediately upstream of said second type IIs restriction site such that said cleavage site of said second type IIs restriction endonuclease is upstream of the fourth restriction site.

20. The vector of claim 19 further including a third type IIs restriction site and a fourth type IIs restriction site, the third type IIs restriction site being positioned upstream of said first type IIs restriction site such that a third type IIs restriction endonuclease recognizing the third type IIs restriction site has a cleavage site identical to that of said third restriction site, and the fourth type IIs restriction site being positioned downstream of said second type IIs restriction site such that a fourth type IIs restriction endonuclease recognizing the fourth type IIs restriction site has a cleavage site identical to that of said fourth restriction site.

21. A method of analyzing gene expression in a cell or tissue, the method comprising the steps of:

(a) forming a population of cDNA molecules from mRNA of a cell or tissue;

(b) determining the nucleotide sequence of a portion of each end of each cDNA molecule of the population so that a pair of nucleotide sequences is obtained for each cDNA of the population; and

(c) tabulating the pairs of nucleotide sequences to form a frequency distribution of gene expression in the cell or tissue.

22. The method of claim 21 wherein said step of determining said nucleotide sequence of said end of each restriction fragment includes the steps of enzymatically removing a segment of nucleotides from each said end;

ligating the segment of nucleotides from each said end together to form a pair of segments, ligating a sample of pairs of segments from said population of cDNA molecules to form one or more concatenations of pairs of segments, and sequencing the concatenations of pairs of segments.