Constructing Contigs from Large‐Insert Clones

Simon Foote1, Vikki Marshall1, David J. Munroe2, Julia A. Segre3

1 Walter and Eliza Hall Institute, Victoria, Australia, 2 Sequana Therapeutics, LaJolla, California, 3 Howard Hughes Medical Institute, Chicago, Illinois
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.10
DOI:  10.1002/0471142905.hg0510s15
Online Posting Date:  May, 2001
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Abstract

This unit describes three approaches that are widely used to define alignments between overlapping clones bearing large‐insert genomic DNA and to generate extensive contiguous overlapping sets of clones (contigs). The three approaches are sequence‐tagged site (STS) content mapping, repetitive‐element hybridization fingerprinting, and Alu‐PCR fingerprinting. Methods for isolating the necessary BAC DNA suitable for automated fluorescent sequencing and generating new STS markers are discussed in support protocols. An alternate protocol presents repetitive‐element hybridization fingerprinting to detect overlaps and build contigs with full‐genomic YAC libraries.

     
 
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Table of Contents

  • Basic Protocol 1: STS Content Mapping
  • Basic Protocol 2: Constructing Contigs of Subgenomic Regions—STS Content Mapping Supplemented with Clone Walking
  • Basic Protocol 3: BAC End Sequencing for Generating a BAC Contig
  • Support Protocol 1: Purification of Sequencing‐Quality BAC DNA by Alkaline Lysis and Cesium Chloride Gradient
  • Support Protocol 2: Purification of Sequencing‐Quality BAC DNA by Peg Precipitation
  • Support Protocol 3: Purification of Sequencing‐Quality BAC DNA with Ion‐Exchange Resins
  • Alternate Protocol 1: Repetitive‐Element Hybridization Fingerprinting
  • Support Protocol 4: Preparation of Adenovirus‐2 DNA Molecular Size Standards
  • Support Protocol 5: Alu‐PCR Fingerprinting
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: STS Content Mapping

  Materials
  • BAC DNA (see protocol 4, protocol 52, or protocol 63)
  • 0.8 pmol/µl T7 primer: 5′‐TAA TAC GAC TCA CTA TAG GG‐3′
  • 0.8 pmol/µl SP6 primer: 5′‐AGC TAT TTA GGT GAC ACT ATA G‐3′
  • Prism DyeDeoxy terminator Ready Reaction Mix kit (Perkin‐Elmer)
  • Sequencing precipitation solution: 0.5 mM MgCl 2/70% ethanol
  • recipeFormamide loading buffer (see recipe)
  • 0.2‐ml thin‐walled tubes
  • Model 9600 thermal cycler (Perkin‐Elmer) or equivalent
  • Automated sequencer (Perkin‐Elmer)
  • Additional reagents and equipment for quantitating DNA ( appendix 3D) and denaturing PAGE ( appendix 3F)

Basic Protocol 2: Constructing Contigs of Subgenomic Regions—STS Content Mapping Supplemented with Clone Walking

  Materials
  • BAC clone of interest in E. coli host
  • CAP plates: recipeLG agar plates (see recipe) containing 12.5 µg/ml chloramphenicol
  • 10 µM STS‐specific oligonucleotide primer
  • LB medium ( appendix 2D) containing 12.5 µg/ml chloramphenicol
  • Glycerol, sterile
  • recipeLysis solution I (see recipe)
  • Lysozyme
  • recipeLysis solution II (see recipe)
  • recipeLysis solution III (see recipe)
  • Isopropanol (propan‐2‐ol; isopropyl alcohol)
  • TE buffer, pH 8.5 ( appendix 2D)
  • CsCl
  • 10 mg/ml ethidium bromide solution ( appendix 2D)
  • Isoamyl alcohol
  • Absolute and 70% ethanol
  • 96‐well deep‐well microtiter plate (Beckman)
  • Microcentrifuge tubes, sterile
  • 500‐ml 2× dry‐spin bottles, autoclaved
  • Sorvall centrifuge and GS‐3 rotor (or equivalent)
  • Gauze, sterile
  • 10‐ml tubes
  • TLA 100.3 centrifuge tubes (Beckman)
  • Quickseal sealer (Beckman)
  • TLA 100 mini‐ultracentrifuge and TLN rotor (Beckman; or equivalent)
  • 20‐G needle
  • 1‐ml syringe with 20‐ or 22‐G needle
  • Additional reagents and equipment for PCR (e.g., unit 5.5) and quantitating DNA ( appendix 3D)

Basic Protocol 3: BAC End Sequencing for Generating a BAC Contig

  Materials
  • TE buffer, pH 8.5 ( appendix 2D)
  • RNase A
  • 13% (w/v) PEG 8000/1.6 M NaCl
  • 50:50 (v/v) phenol/chloroform
  • Chloroform
  • 10 M ammonium acetate ( appendix 2D)
  • Absolute and 70% ethanol
  • Additional reagents and equipment for preparing BAC DNA (see protocol 4, steps to ) and quantitating DNA ( appendix 3D)

Support Protocol 1: Purification of Sequencing‐Quality BAC DNA by Alkaline Lysis and Cesium Chloride Gradient

  Materials
  • recipeQBT buffer (see recipe)
  • recipeQC buffer (see recipe)
  • recipeQF buffer (see recipe)
  • Isopropanol
  • Absolute and 70% ethanol
  • TE buffer, pH 8.5 ( appendix 2D)
  • 3 M sodium acetate, pH 5.3 ( appendix 2D)
  • Qiagen 2500 column
  • 30‐ml Corex centrifuge tubes
  • Sorvall RC5C centrifuge and SS‐34 rotor or equivalent
  • Additional reagents and equipment for preparing BAC DNA (see protocol 4, steps to ) and quantitating DNA ( appendix 3D)

Support Protocol 2: Purification of Sequencing‐Quality BAC DNA by Peg Precipitation

  Materials
  • YAC‐containing yeast clones of interest
  • AHC medium (unit 5.5)
  • Appropriate restriction endonucleases and buffers
  • Adenovirus‐2 DNA molecular size standard mixture (see protocol 8)
  • SeaKem ME agarose (FMC Bioproducts)
  • 0.5× TBE buffer ( appendix 2D)
  • Adenovirus‐2 DNA (Life Technologies)
  • Adenovirus‐2 forward and reverse oligonucleotide PCR primers (Table 5.10.1)
    Table 5.0.1   Materials   Oligonucleotides Used in Hybridization Fingerprinting a   Oligonucleotides Used in Hybridization Fingerprinting   Hybridization and Washing Conditions for Oligonucleotide Probes Used in Repetitive‐Element Hybridization Fingerprinting d   Hybridization and Washing Conditions for Oligonucleotide Probes Used in Repetitive‐Element Hybridization Fingerprinting

    Oligonucleotide Sequence
    Alu‐1 5′‐TGA GC(CT) (GA)(AT)G AT(CT) (GA)(CT)(GA)CCA  (CT)TG CAC TCC AGC CTG GG‐3′
    Alu‐2 5′‐GCC TCC CAA AGT GCT GGG AATT ACA GG(CT)  (GA)TG AGC CA‐3′
    L1 5′‐TGG GTG CAG C(AG)C ACC A(AG)C ATG GCA  CAT GGT ATA CAT ATG TAA C(AT)A ACC TGC AC‐C′‐3′
    Single‐sequence repeat 5′‐(AG) 12‐3′
    5′‐(AAGT) 8‐3′
    5′‐(AATT) 8‐3′
    5′‐(ACAG) 8‐3′
    5′‐(ACAT) 8‐3′
    5′‐(ACTC) 8‐3′
    5′‐(ACTG) 8‐3′
    5′‐(AGAT) 8‐3′
    5′‐(AGGG) 8‐3′
    Adenovirus forward b 5′‐TAC GGG AAG TGA CAA TTT TCG‐3′
    Adenovirus reverse c 5′‐TAA ATA CAC TGC GCG TCA GC‐3′
    Oligonucleotide e T m (°C) T hyb (°C) T wash (°C) Wash solution Wash times (min)
    Alu‐1 ND 65 65 2× SSC/0.1% SDS 20; 20; 20
    Alu‐2 ND 65 65 2× SSC/0.1% SDS 20; 20; 20
    L1 ND 65 42 6× SSC/0.1% SDS 20; 20; 20
    (AG) 12 54 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (AACT) 8 51.4 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (AAGT) 8 51.4 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (AATT) 8 51.5 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACAG) 8 69.2 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACAT) 8 55.1 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACTC) 8 68.5 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACTG) 8 69.2 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (AGAT) 8 50.8 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (AGGG) 8 83.7 65 65 5× SSC/0.1% SDS 1; 5; 10; 1

     aFor details of oligonucleotide synthesis and purification, see CPMB UNITS & .
     bMelting temperature = 60°C; starts at nucleotide 195 in complete genome sequence.
     cMelting temperature = 60°C; ends at nucleotide 472 in complete genome sequence.
  • 2 mM 4dNTP mix ( appendix 2D)
  • 10× PCR amplification buffer ( appendix 2D)
  • Taq DNA polymerase
  • Mineral oil
  • 10 M ammonium acetate ( appendix 2D)
  • 100% ethanol, ice cold
  • Church's hybridization buffer (unit 2.3), 65°C
  • India ink containing 0.5 µCi/ml 32P
  • 0.4 N NaOH
  • recipeModerate stripping solution (see recipe)
  • Alu, L1, or simple sequence repeat oligonucleotide DNA for probes (Table 5.10.1)
  • Washing solution of appropriate stringency for probe (Table 5.10.2)
    Table 5.0.2   Materials   Oligonucleotides Used in Hybridization Fingerprinting a   Oligonucleotides Used in Hybridization Fingerprinting   Hybridization and Washing Conditions for Oligonucleotide Probes Used in Repetitive‐Element Hybridization Fingerprinting d   Hybridization and Washing Conditions for Oligonucleotide Probes Used in Repetitive‐Element Hybridization Fingerprinting

    Oligonucleotide Sequence
    Alu‐1 5′‐TGA GC(CT) (GA)(AT)G AT(CT) (GA)(CT)(GA)CCA  (CT)TG CAC TCC AGC CTG GG‐3′
    Alu‐2 5′‐GCC TCC CAA AGT GCT GGG AATT ACA GG(CT)  (GA)TG AGC CA‐3′
    L1 5′‐TGG GTG CAG C(AG)C ACC A(AG)C ATG GCA  CAT GGT ATA CAT ATG TAA C(AT)A ACC TGC AC‐C′‐3′
    Single‐sequence repeat 5′‐(AG) 12‐3′
    5′‐(AAGT) 8‐3′
    5′‐(AATT) 8‐3′
    5′‐(ACAG) 8‐3′
    5′‐(ACAT) 8‐3′
    5′‐(ACTC) 8‐3′
    5′‐(ACTG) 8‐3′
    5′‐(AGAT) 8‐3′
    5′‐(AGGG) 8‐3′
    Adenovirus forward b 5′‐TAC GGG AAG TGA CAA TTT TCG‐3′
    Adenovirus reverse c 5′‐TAA ATA CAC TGC GCG TCA GC‐3′
    Oligonucleotide e T m (°C) T hyb (°C) T wash (°C) Wash solution Wash times (min)
    Alu‐1 ND 65 65 2× SSC/0.1% SDS 20; 20; 20
    Alu‐2 ND 65 65 2× SSC/0.1% SDS 20; 20; 20
    L1 ND 65 42 6× SSC/0.1% SDS 20; 20; 20
    (AG) 12 54 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (AACT) 8 51.4 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (AAGT) 8 51.4 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (AATT) 8 51.5 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACAG) 8 69.2 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACAT) 8 55.1 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACTC) 8 68.5 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (ACTG) 8 69.2 65 65 5× SSC/0.1% SDS 1; 5; 10; 1
    (AGAT) 8 50.8 50 50 5× SSC/0.1% SDS 1; 5; 10; 1
    (AGGG) 8 83.7 65 65 5× SSC/0.1% SDS 1; 5; 10; 1

     dAbbreviations: T m, melting temperature; T hyb, hybridization temperature; T wash, washing temperature; ND, not determined.
     eSee Table 5.10.1 for additional information about oligonucleotide probes.
  • Water baths at 65°C and other appropriate temperatures (Table 5.10.2)
  • Hole punch
  • Ink pad and stamp
  • Additional reagents and equipment for preparation of yeast DNA (unit 5.9), DNA quantitation by fluorimetry ( appendix 3D), agarose gel electrophoresis (unit 2.7), Southern blotting and hybridization (unit 2.7), ethanol precipitation of DNA ( appendix 3C), and preparation of radiolabeled probes ( appendix 3E)

Support Protocol 3: Purification of Sequencing‐Quality BAC DNA with Ion‐Exchange Resins

  Materials
  • Adenovirus‐2 DNA (Life Technologies)
  • Restriction endonucleases (Table 5.10.3) and appropriate buffers
    Table 5.0.3   Materials   Restriction Enzymes Used to Cut Adenovirus‐2 DNA for Molecular‐Size‐Standard Mixture and Sizes of Bands Revealed by Hybridization f   Restriction Enzymes Used to Cut Adenovirus‐2 DNA for Molecular‐Size‐Standard Mixture and Sizes of Bands Revealed by Hybridization

    Enzyme Band size (bp) Enzyme Band size (bp)
    BstXI 608 StuI  3151
    NaeI 810 AflII  3527
    SmaI 1006 BclI  4056
    DraI 1193 ApaI  4609
    PflMI 1461 PmlI  5179
    EcoNI 1707 XhoI  5778
    BsteII 1912 EagI  6493
    NcoI 2200 FspI  8088
    TthIII 2389 BamHI 10,680
    HindIII 2798 SfiI 16,300

     fThe result of hybridization of the adenovirus‐2 size standard mixture to an adenovirus‐2 probe is shown in Figure .
  • TE buffer ( appendix 2D)
  • recipe5× BGE loading buffer (see recipe)
  • Additional reagents and equipment for phenol/chloroform extraction and ethanol precipitation ( appendix 3C)

Alternate Protocol 1: Repetitive‐Element Hybridization Fingerprinting

  Materials
  • Yeast DNA from YAC‐bearing clones
  • 2.5 mM Tris⋅Cl, pH 8.0 ( appendix 2D)
  • Alu primers: 20 µM Alu‐S/Alu‐J mixture (4:1 w/w), 20 µM Alu‐end, and 20 µM 47‐23 ( .)
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D) and 0.5% (w/v) Nonidet P‐40 (NP‐40)
  • Klenow fragment of DNA polymerase I
  • Chloroform
  • 3 M sodium acetate, pH 5.2 ( appendix 2D)
  • Isopropanol
  • 70% ethanol, ice cold
  • TE buffer ( appendix 2D)
  • DNA molecular size standards (e.g., 123‐bp ladder)
  • 0.5‐ml microcentrifuge tubes
  • 65°C water bath
  • Additional reagents and equipment for preparation of yeast DNA in agarose blocks (unit 5.1) or by miniprep procedure (unit 5.9), inter‐IRS PCR (unit 5.9), quantitation of DNA ( appendix 3D), phenol/chloroform extraction and alcohol precipitation ( appendix 3C), and agarose gel electrophoresis (unit 2.7)
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Figures

Videos

Literature Cited

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Key References
   Green et al., 1991b. See above.
  First description of the application of STS‐based YAC mapping.
   Olson et al., 1989. See above.
  Description of STS concept.
   Nelson et al., 1991. See above.
  First paper demonstrating Alu‐PCR fingerprinting of YAC clones.
   Bellanne‐Chantelot et al., 1992. See above.
  Describes assembly of 1000 contigs by fingerprinting of 22,000 human YACs.
   Foote et al., 1992. See above.
  Describes assembly of a complete map of the human Y chromosome using STS to anchor contigs and YAC fingerprinting to close gaps with undetected overlaps.
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