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Construction of Chromosome Jumping and Linking Libraries in E. coli

Mitchell L. Drumm1

1Case Western Reserve University, Cleveland, Ohio

Publication Name: 
Current Protocols in Human Genetics
Unit Number: 
Unit 5.4
DOI: 
10.1002/0471142905.hg0504s01
Online Posting Date: 
May, 2001
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Abstract

Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant point on the same chromosome without cloning the intervening sequences as in chromosome walking. A linking library consists of genomic fragments that each contain a particular restriction site with a marker inserted into it. Because the sequences flanking the marker represent the termini of two contiguous genomic restriction fragments, the linking clones can be used, in conjunction with a specific jumping library made with the same restriction enzyme, to expedite jumping between consecutive restriction fragments. Separate protocols present strategies for constructing general and specific jumping libraries from total genomic DNA. An additional protocol details construction of a linking library from flow-sorted chromosomes. Support protocols provide instructions for preparing genomic insert DNA, supF gene fragments, and Chromosome jumping allows the use of one point on a chromosome as a starting point for exploring another potentially distant.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Construction of a General Jumping Library from Total Genomic DNA
  • Alternate Protocol: Construction of a Specific Jumping Library from Total Genomic DNA
  • Basic Protocol 2: Construction of a Linking Library from Flow-Sorted Chromosomes
  • Support Protocol 1: Preparation and Testing of Genomic Insert DNA Embedded in Agarose Blocks
  • Support Protocol 2: Preparation and Testing of supF Gene Fragment
  • Support Protocol 3: Preparation of Bacteriophage Vector DNA
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Construction of a General Jumping Library from Total Genomic DNA

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D for suppliers, see suppliers appendix
  • 50-µl agarose blocks containing high-molecular-weight genomic DNA (first support protocol)
  • MboI restriction endonuclease and 10× buffer
  • 0.5 M EDTA
  • -concatemer DNA molecular size standards in agarose blocks (unit 5.1)
  • 0.5 µg/ml ethidium bromide solution
  • 0.5× TBE buffer (appendix 2)
  • Sucrose powder or crystals
  • TE buffer, pH 7.5
  • 200 U/µl T4 DNA ligase (measured in cohesive-end units)
  • 10× ligase buffer: 500 mM Tris×Cl (pH 7.5)/10 mM MgCl2
  • supF DNA excised with BamHI (second support protocol)
  • 100 mM ATP, pH 7.2
  • 3 M sodium acetate, pH 5.2
  • 95% (ice-cold) and 70% ethanol
  • Yeast tRNA (Sigma)
  • 2 mg/ml DNase-free RNase A (appendix 2)
  • 20 U/µl EcoRI restriction endonuclease and 10× buffer
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (appendix 3C)
  • Chloroform
  • EcoRI-digested Charon3AlacXba or HindIII-digested Charon 21A vector DNA (third support protocol; see Fig. 5.4.2)
  • Selective host: E. coli strain MC1061 (ATCC #53338; cpmb Table 1.4.5) or equivalent
  • Permissive host: E. coli strain LE392 (ATCC #33572; cpmb Table 1.4.5) or equivalent
  • Sterile dialysis tubing (cpmb appendix 3)
  • Beckman SW-28 rotor and SW-28 centrifuge tubes (or equivalent)
  • 14°C water bath
  • Additional reagents and equipment for pulsed-field gel electrophoresis (unit 5.1), electroelution from agarose gel (cpmb unit 2.6),spectrophotometric quantitation of DNA (appendix 3D), phenol extraction and ethanol precipitation (appendix 3C),agarose gel electrophoresis (unit 2.7), DNA labeling (appendix 3E), Southern blotting and hybridization (unit 2.7) and packaging, plating, and screening of bacteriophage libraries (units 2.2, 2.3 & cpmb unit 1.11)

CAUTION: Phenol and chloroform are hazardous; see appendix 2A for guidelines handling, storage, and disposal.

Alternate Protocol: Construction of a Specific Jumping Library from Total Genomic DNA

 Additional Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • NotI restriction endonuclease and 10× buffer
  • Block digestion buffer (see recipe)
  • 1 mM phenylmethylsulfonylfluoride(PMSF; unit 5.1)in TE buffer, pH 7.5 (prepare fresh)
  • 50 mM Tris×Cl (pH 7.6)/10 mM MgCl2
  • supF DNA excised with NotI (second support protocol)
  • 50° and 65°C water baths
  • Additional reagents and equipment for pulsed-field gel electrophoresis (unit 5.1)

CAUTION: PMSF is hazardous; see appendix 2A for guidelines on handling, storage, and disposal.

Basic Protocol 2: Construction of a Linking Library from Flow-Sorted Chromosomes

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Flow-sorted chromosomes containing genomic DNA of interest (Fuscoe et al., 1986)
  • Chromosome cleanup buffer (see recipe)
  • 25:24:1 (v/v/v) phenol/chloroform/isoamyl alcohol (appendix 3C)
  • Chloroform
  • 3 M sodium acetate, pH 5.2
  • 95% (ice-cold) and 70% ethanol
  • TE buffer, pH 7.5
  • BglII and NotI restriction endonucleases and 10× buffers
  • 200 U/µl T4 DNA ligase (measured in cohesive-end units)
  • 10× ligase buffer: 500 mM Tris×Cl (pH 7.5)/10 MgCl2
  • supF DNA excised with BamHI (Support Protocol 2)
  • NotI-digested Charon 3AlacXN vector DNA or other vector with unique rare-cutting restriction site (first support protocol)
  • Beckman SW-60 rotor (or equivalent)
  • 14°C and 55° water baths
  • Additional reagents and equipment for phenol extraction and ethanol precipitation of DNA (appendix 3C) and packaging, plating, and screening of bacteriophage libraries (units 2.2-2.7 & cpmb unit 1.11)

CAUTION: Phenol and chloroform are hazardous; see appendix 2A for guidelines on handling, storage, and disposal .

Support Protocol 1: Preparation and Testing of Genomic Insert DNA Embedded in Agarose Blocks

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • Source cells
  • TE buffer, pH 7.5
  • MboI restriction endonuclease and 10× buffer
  • Additional reagents and equipment for preparing genomic DNA in agarose blocks and PFGE (unit 5.1), and agarose gel electrophoresis (unit 2.7)

Support Protocol 2: Preparation and Testing of supF Gene Fragment

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D, for suppliers, see suppliers appendix
  • pTBsupFor pBXN supF DNA (available from F.S. Collins, National Center for Human Genome Research, Bethesda, Md.)
  • BamHI or NotI restriction endonuclease and appropriate 10× buffer
  • TE buffer, pH 7.5
  • 200 U/µl T4 DNA ligase (measured in cohesive-end units)
  • 10× ligase buffer: 500 mM Tris×Cl (pH 7.5)/10 mM MgCl2
  • 14°C water bath
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), electroelution from agarose gel (cpmb unit 2.6), and spectrophotometric quantitation of DNA (appendix 3D)

Support Protocol 3: Preparation of Bacteriophage Vector DNA

 Materials
For recipes, see Reagents and Solutions in this unit (or cross-referenced unit); for common stock solutions, see appendix 2D; for suppliers, see suppliers appendix.
  • phage vector DNA: 0-insert, amber-mutated phage with unique cloning site, e.g., Charon 3AlacXba, Charon 3AlacXN, or Charon 21A (Fig. 5.4.2)
  • Appropriate restriction endonuclease (e.g., EcoRI, HindIII, or NotI) and 10× buffer
  • TE buffer, pH 7.5
  • 65°C water bath

Additional reagents and equipment for agarose gel electrophoresis (unit 2.7), and phenol extraction, and ethanol precipitation (appendix 3C)

Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 5.4.1
    Comparison of jumping and linking clones. (A) Comparison of the strategies for constructing general and specific jumping libraries and linking libraries. In each, genomic DNA is digested (I) to generate termini compatible with the supF selectable marker. The digested fragments (II) are ligated with the supF gene fragment (III), thereby generating circular molecules. The circles are then cleaved with an enzyme compatible with the cloning vector (IV), cloned into the vector, packaged, and transfected into a host that permits phage growth only if supF is present (V). (B) shows the type of fragments cloned in each of the three types of libraries. A general jumping fragment is shown by the jump from A to B. General jumping fragments can potentially be achieved at any point on a chromosome, but a complex library of such fragments is required to represent the genome. A specific jumping fragment is shown by the jump from C to D. Specific jumping libraries are only accessible from rare-cutter restriction sites, but a relatively small number of such clones is needed to represent the entire genome. Specific jumping libraries are particularly powerful when used in conjunction with linking libraries. A linking library contains clones of sequences on both sides of rare restriction sites such as D and E. After jumping from C to D, one can continue to jump to consecutive restriction sites by using D as a probe to isolate the D/E linking clone, then using E as a probe to jump to the next NotI site, and so on.

    View Image
  • Figure 5.4.2
    Examples of phage vectors suitable for jumping and linking libraries. Charon 3AlacXba and Charon3AlacXN are available from Dr. F.S. Collins, National Center for Human Genome Research, Bethesda, Md., and Charon 21A is obtainable from Dr. F. Blattner, University of Wisconsin. These vectors each contain two amber mutations (Wam, Eam, Aam, or Bam) in the phage left arm, allowing selection by supF. Unique cloning sites separate the left and right arms. Positions in kilobases are shown in parenthesis and cloning capacity is shown at right.

    View Image

Literature Cited

Literature Cited
    Collins, F.S., Drumm, M.L., Cole, J.L., Lockwood, W.K., Vande Woude, G.F., and Ianuzzi, M.C. 1987. Construction of a general human chromosome jumping library, with application to cystic fibrosis. Science 235:1046-1049.
    Fuscoe, J.C., Clark, L.M., and Van Dilla, M.A. 1986. Construction of fifteen human chromosome-specific DNA libraries from flow purified chromosomes. Cytogenet. Cell. Genet. 43:79-86.
    Poustka, A., Pohl, T.M., Barlow, D.P., Frischauf, A.-M., and Lehrach, H. 1987. Construction and use of human chromosome jumping libraries from NotI-digested DNA. Nature 325:353-355.
    Wallace, M.R., Fountain, J.W., Brereton, A.M., and Collins, F.S. 1989. Direct construction of a chromosome-specific NotI linking library from flow-sorted chromosomes. Nucl. Acids Res. 17:1665-1677.
     
 
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