Screening Large‐Insert Libraries by Hybridization

Mark T. Ross (gridding: YAC, BAC, and PAC colony hybridization)1, Sam LaBrie (BAC/PAC colony hybridization and analysis)2, John McPherson (overgo probes and hybridization)3, Vincent P. Stanton4

1 The Sanger Centre Wellcome Trust Genome Campus, Hixton, Cambridge, UK, 2 Genome Systems, St. Louis, Missouri, 3 Washington University School of Medicine, St. Louis, Missouri, 4 Variagenic, Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 5.6
DOI:  10.1002/0471142905.hg0506s21
Online Posting Date:  May, 2001
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Abstract

Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR‐product hybridization. The first protocol describes methods for arraying large‐insert clones. Subsequent protocols discuss hybridizing YAC and BAC/PAC colony blots. An alternate protocol provides a guide for preparing and hybridizing with, high‐specific‐activity oligonucleotide probes called “overgo” probes. Support protocols indicate the procedures for identifying the plate and well address of positive clones by manual and software‐assisted methods. Hybridization of Alu‐PCR‐product probes to Alu‐PCR products of YAC clones are provided. An alternate protocol describes a method for preparing DNA from individual YACs and for producing and using Alu‐PCR product blots from individual YACs. Additional support protocols provide guidelines for preparing Alu‐PCR probes from cosmids, YACs, BACs/PACs, and somatic cell hybrids, and describe the use of 96‐well plates to radiolabel and spin‐column‐purify large numbers of probes. A method for YAC walking is also provided.Three types of hybridization screening are discussed in this unit colony hybridization, DNA hybridization, and YAC Alu‐PCR.

     
 
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Table of Contents

  • Basic Protocol 1: Arraying Colonies and DNA at Low and High Density
  • Support Protocol 1: Preparing YAC Colony Blots for Hybridization
  • Support Protocol 2: Preparing BAC and PAC Colony Blots for Hybridization
  • Basic Protocol 2: Screening YAC Libraries by Colony Hybridization
  • Basic Protocol 3: Screening BAC, PAC, and P1 Libraries by Colony Hybridization
  • Alternate Protocol 1: Preparing and Hybridizing Colony Blots with Overgo Oligonucleotide Probes
  • Support Protocol 3: Identifying the Plate and Well Addresses of Positives on Autoradiograph Films
  • Support Protocol 4: Analyzing Hybridization Results Using Phosphorimaging and ArrayVision Software
  • Basic Protocol 4: Screening YAC Libraries by Hybridization of Alu‐PCR Products
  • Alternate Protocol 2: Screening Alu‐PCR Product Blots of Individual YACs
  • Support Protocol 5: Generating Alu‐PCR Probes from Large‐Insert Clones
  • Support Protocol 6: Radiolabeling and Purification of DNA Probes in 96‐Well Microtiter Plates
  • Support Protocol 7: YAC Walking with Alu‐PCR Product Blots
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Arraying Colonies and DNA at Low and High Density

  Materials
  • YAC colonies arrayed on nylon filters (see protocol 1)
  • YPD agar plates (unit 5.5) made in 8 × 12–cm (Genomic Solutions) or 22 × 22–cm plates (Nunc)
  • recipeSpheroplasting solution (see recipe)
  • Denaturing solution A: 1.5 M NaCl/0.5 M NaOH
  • Neutralizing solution A: 0.5 M Tris˙Cl (pH 7.4)/1.5 M NaCl
  • 0.1× neutralizing solution A: 0.05 M Tris˙Cl (pH 7.4)/0.15 M NaCl
  • 0.25 mg/ml proteinase K in 0.1× neutralizing solution A, 37°C
  • 50 mM Tris˙Cl (pH 7.4; appendix 2D)
  • 30° and 37°C incubators
  • Whatman 3MM filter paper
  • Plastic trays
  • Orbital shaker

Support Protocol 1: Preparing YAC Colony Blots for Hybridization

  Materials
  • PAC or BAC colonies arrayed on nylon filters (see protocol 1)
  • LB agar plates containing 30 µg/ml kanamycin (PACs) or 20 µg/ml chloramphenicol (BACs) made in 8 × 12–cm (Genomic Solutions) or 22 × 22–cm plates (Nunc)
  • 10% sodium dodecyl sulfate (SDS; appendix 2D)
  • Denaturing solution A: 0.5 N NaOH/1.5 M NaCl
  • Neutralizing solution A: 0.5 M Tris⋅Cl (pH 7.4)/1.5 M NaCl
  • 0.1× neutralizing solution A: 0.05 M Tris⋅Cl (pH 7.4)/0.15 M NaCl
  • SSC/0.1% SDS
  • SSC ( appendix 2D)
  • 50 mM Tris⋅Cl (pH 7.4; appendix 2D)
  • 37°C incubator
  • Whatman 3MM filter paper
  • Plastic trays
  • Orbital shaker

Support Protocol 2: Preparing BAC and PAC Colony Blots for Hybridization

  Materials
  • 50 ng hybridization probe DNA (see Commentary) or PCR product (e.g., STS) separated in agarose gel (see step )
  • 10 to 40 µCi [α‐32P]dNTP (3000 Ci/mmol)
  • 50 ng S. cerevisiae genomic DNA from YAC host S. cerevisiae strain (unit 5.9; see Commentary; optional)
  • 25 µCi [α‐35S]dNTP (600 Ci/mmol; optional)
  • 10× PCR amplification buffer with 15 mM MgCl 2 ( appendix 2D), prepared without gelatin (optional)
  • 2 mM 3dNTP mix (dATP/dGTP/dTTP; appendix 2D; optional)
  • 5 U/µl Taq DNA polymerase (optional)
  • Primer mixture appropriate to amplify the agarose‐excised PCR product (100 ng/µl each; see step ; optional)
  • YAC colonies arrayed on nylon filters, prepared for hybridization (see protocol 2)
  • recipeHybridization solution A (see recipe), prewarmed to 65°C and filtered using a 0.45‐µm filter
  • 20×, 2×, and 0.5× SSC ( appendix 2D)
  • 10 mg/ml sheared human genomic DNA (average size 200 to 500 bp; optional)
  • Wash solution: 0.5× SSC/1% (w/v) N‐lauroylsarcosine, room temperature and 65°C (see recipe for recipeN‐lauroylsarcosine )
  • 65°C oven
  • 100°C water bath
  • Plastic boxes or glass pans
  • Autoradiography equipment and supplies
  • Additional reagents and equipment for labeling by random oligonucleotide–primed synthesis ( appendix 3E)

Basic Protocol 2: Screening YAC Libraries by Colony Hybridization

  Materials
  • High‐density filters for a BAC, PAC, or P1 library (commercially available, or prepared using protocol 1 and protocol 3)
  • 50 ng DNA for radiolabeling reaction (free of any vector sequences)
  • 10 to 40 µCi [α‐32P]dNTP (3000 Ci/mmol)
  • recipePrehybridization solution (see recipe)
  • recipeHybridization solution B (see recipe)
  • 10 mg/ml sonicated human genomic DNA (optional)
  • Wash solution 1: 1 mM Tris⋅Cl, pH 8.0 ( appendix 2D)/1% recipeN‐lauroylsarcosine (see recipe), prepared fresh daily
  • Wash solution 2: 1 mM Tris⋅Cl, pH 8.0 (store at room temperature)
  • Paper towels
  • Backing film (old autoradiographs)
  • Autoradiography film
  • Static Guard (anti‐static spray)
  • Saran Wrap or other UV‐transparent plastic wrap
  • Hybridization oven
  • Hybridization tubes
  • 100°C water bath or incubator
  • Autoradiography equipment
  • Additional reagents and equipment for labeling by random oligonucleotide–primed synthesis ( appendix 3E)

Basic Protocol 3: Screening BAC, PAC, and P1 Libraries by Colony Hybridization

  Materials
  • Two appropriate overlapping 24‐mer oligonucleotides for generating each overgo probe
  • 2 mg/ml bovine serum albumin (BSA); appendix 2D
  • recipeOvergo labeling buffer (see recipe)
  • 10 µCi/µl [α‐32P]dATP (∼3000 Ci/mmol)
  • 10 µCi/µl [α‐32P]dCTP (∼3000 Ci/mmol)
  • Klenow fragment of E. coli DNA polymerase I
  • 10% (w/v) trichoroacetic acid (TCA)
  • 95% ethanol
  • Church's hybridization buffer (unit 2.3), 60°C
  • 20× SSC ( appendix 2D)
  • 10% SDS ( appendix 2D)
  • Target DNA immobilized on nylon colony blot filters (see protocol 1 and Support Protocols protocol 21 or protocol 32)
  • Glass microfiber filter disk (A/E fiber filter; Gelman)
  • Vacuum filter holder
  • Sephadex G‐50 microspin column (e.g., NICK Spin Columns; Amersham Pharmacia Biotech)
  • 60°C hybridization oven
  • Autoradiography equipment and supplies

Alternate Protocol 1: Preparing and Hybridizing Colony Blots with Overgo Oligonucleotide Probes

  Materials
  • Phosphor screens (one per filter in the hybridization set)
  • Phosphorimager with large enough imaging screen for 22 × 22–cm filters
  • ArrayVision (Imaging Research; distributed by Genome Systems, Molecular Dynamics, and Clontech)
  • Computer hardware: Windows NT system with >200 MHz processor speed, >100 MB RAM, and (preferably) 20‐in. monitor

Support Protocol 3: Identifying the Plate and Well Addresses of Positives on Autoradiograph Films

  Materials
  • 100 µg/ml YAC pool DNA prepared in agarose blocks (containing ≤100 Mb insert DNA; units 5.5 & 5.1)
  • 0.25× TE buffer: 2.5 mM Tris⋅Cl (pH 8.0)/0.25 mM EDTA
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 20 µM oligonucleotide primers 47‐23, Alu‐S + Alu‐J, and Alu‐end (see unit 5.9 for sequence)
  • 5 U/µl Taq DNA polymerase or other thermostable DNA polymerase
  • India ink
  • 0.5 M EDTA, pH 8.0 ( appendix 2D)
  • Denaturing solution B: 0.4 M NaOH/0.6 M NaCl
  • Neutralizing solution B: 0.5 M Tris⋅Cl (pH 7.5)/0.5 M NaCl
  • recipe5× and 3× SSPE (see recipe)
  • Wash solution A: 0.1× SSC/0.1% (w/v) SDS
  • Alu‐PCR generated probes (see protocol 11)
  • Low gelling/melting temperature agarose (e.g., SeaPlaque GTG agarose, FMC Bioproducts)
  • 10 mg/ml sheared human genomic DNA (average size 200 to 500 bp)
  • 20× and 0.1× SSC ( appendix 2D)
  • Mineral oil
  • recipePrehybridization/hybridization solution (see recipe)
  • Wash solution B: 1× SSC/0.5% (w/v) SDS
  • Wash solution C: 0.1× SSC/0.25% (w/v) SDS, 65°C
  • 65° and 100°C water baths
  • 96‐ or 384‐well microtiter plates
  • Thermal cycler
  • 96‐pin replicator suitable for producing high‐density arrays from 96‐well microtiter plates (e.g., manual device from Washington University Machine Shop or Beckman Biomek 1000)
  • Positively charged nylon membrane (e.g., GeneScreen Plus, Du Pont NEN, or Hybond N+, Amersham)
  • 80°C vacuum oven
  • Gel blot paper (Schleicher and Schuell) or Whatman 3MM filter paper
  • 42° and 65°C incubators
  • Autoradiography equipment and supplies
  • Additional reagents and equipment for preparing YAC DNA in agarose blocks (unit 5.1) or in solution (unit 5.9), construction of DNA pools (unit 5.5), agarose gel electrophoresis (unit 2.7), arraying YAC DNA (see protocol 1), generating Alu‐PCR probes (see protocol 11), and labeling by random oligonucleotide‐primed synthesis and spin‐column chromatography to remove unincorporated nucleotides ( appendix 3E or protocol 12)

Support Protocol 4: Analyzing Hybridization Results Using Phosphorimaging and ArrayVision Software

  • Yeast colonies
  • AHC medium (unit 5.5)
  • recipe40 mg/ml yeast lytic enzyme solution (see recipe)
  • SCEM buffer: SCE buffer (unit 5.1) containing 2 µg/ml 2‐ME (added just before use)
  • 0.5% (w/v) SDS
  • TES buffer (see recipe)
  • 5 M potassium acetate solution, pH 4.8 (unit 5.3)
  • Isopropanol
  • 70% ethanol
  • 5 µg/ml DNase‐free RNase A ( appendix 2D) in TE buffer ( appendix 2D)
  • 96‐well microtiter plates with deep, flat‐bottom wells
  • Transtar 96 (Costar) 96‐channel pipettor or other multichannel pipettor
  • Flexible 96‐well microtiter plates with round‐bottom wells (Falcon) or PEC‐9600 plates (Perkin‐Elmer Cetus)
  • Sorvall RT‐6000 centrifuge and H1000B rotor equipped with a microplate carrier (or equivalent)
  • Phase‐contrast microscope

Basic Protocol 4: Screening YAC Libraries by Hybridization of Alu‐PCR Products

  Materials
  • Template DNA: 10 to 100 pg λ, BAC, PAC (unit 5.15), or cosmid DNA; or 1 to 10 ng total yeast DNA from a clone with a YAC, protocol 10, steps or unit 5.9, protocol 10; or 20 to 200 ng total DNA from a somatic cell hybrid (units 3.1 & 3.2) and rodent host cell line
  • 10× PCR amplification buffer containing 15 mM MgCl 2 ( appendix 2D)
  • 1.25 mM 4dNTP mix ( appendix 2D)
  • 20 µM oligonucleotide primers Alu‐S + Alu‐J, Alu‐end, or 47‐23 (see unit 5.9 for sequences)
  • 5 U/µl Taq DNA polymerase
  • Thermal cycler
  • Additional reagents and equipment for Alu‐PCR (unit 5.9), labeling Alu‐PCR probes (see protocol 9), or labeling multiple probes in microtiter plates (see protocol 12); agarose gel electrophoresis (unit 2.7); production of a DNA library (CPMB UNIT ); and Alu‐PCR fingerprinting (unit 5.10)

Alternate Protocol 2: Screening Alu‐PCR Product Blots of Individual YACs

  Materials
  • Probe DNA: 1 to 5 µg/µl in H 2O, TE buffer ( appendix 2D), or molten low gelling/melting temperature agarose containing TAE buffer ( appendix 2D)
  • 10 mg/ml bovine serum albumin (BSA; appendix 2D)
  • recipeOligo labeling buffer (OLB; see recipe)
  • 10 µCi/µl [α‐32P]dCTP (∼3000 Ci/mmol)
  • 5 U/µl Klenow fragment of E. coli DNA polymerase I
  • 425‐ to 600‐µm acid‐washed glass beads (Sigma), recipesilanized (see recipe)
  • Sephadex G‐50 resin (Amersham Pharmacia Biotech) swelled in TE buffer (see appendix 3E for swelling procedure)
  • TE buffer
  • 96‐well thin‐walled polycarbonate V‐bottom microtiter plate suitable for PCR (e.g., Costar Thermal Well plates, USA Scientific or MJ Research) or thin‐walled 0.2‐ml tubes (USA Scientific, Robbins Scientific, or Perkin Elmer) arrayed in 96‐well (8 × 12) format (can be purchased in 8‐ or 12‐tube strips)
  • Precut plastic adhesive sealing tape to fit 96‐well microtiter plates (Costar, USA Scientific, or Sigma)
  • 8‐ or 12‐channel pipettor capable of pipetting 5 µl accurately
  • 95°C air oven or thermal cycler with heated lid, compatible with thin‐walled 96‐well plates or 0.2‐ml tubes arrayed in 96‐well format
  • 96‐well flexible U‐bottom microtiter plate (Falcon Flexible Assay Plate)
  • Styrofoam
  • 25‐G needle
  • Lid from pipet‐tip rack
  • Sorvall RT6000 centrifuge and H1000B rotor with microplate carrier (or equivalent)
  • 96‐well polystyrene V‐bottom microtiter plate (e.g., Costar)
  • Aerosol‐barrier pipet tips
  • Additional reagents and equipment for quantitating DNA by TCA precipitation ( appendix 3E)

Support Protocol 5: Generating Alu‐PCR Probes from Large‐Insert Clones

  Materials
  • Clones from contig of interest
  • Autoradiography equipment and supplies
  • Additional reagents and equipment for preparation of yeast DNA in agarose blocks (unit 5.1) or in solution (unit 5.9), Alu‐PCR (see protocol 11 and unit 5.9), agarose gel electrophoresis (unit 2.7), and screening YAC libraries by Alu‐PCR hybridization (see protocol 4)
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Figures

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Literature Cited

Literature Cited
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   Ross, M.T., Nizetic, D., Nguyen, C., Knights, C., Vatcheva, R., Burden, N., Douglas, C., Zehetner, G., Ward, D.C., Baldini, A., and Lehrach, H. 1992. Selection of a human chromosome 21 enriched YAC sub‐library using a chromosome‐specific composite probe. Nature Genet. 1:284‐290.
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   Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed., pp 1.25‐1.28. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Shizuya, H., Birren, B., Kim, U.J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. 1992. Cloning and stable maintenance of 300‐kilobase‐pair fragments of human DNA in Escherichia coli using an F‐factor‐based vector. Proc. Natl. Acad. Sci. U.S.A. 89:8794‐8797.
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Key References
   Nelson, D.L., Ledbetter, S.A., Corbo, L., Victoria, M.F., Ramirez‐Solis, R., Webster, T.D., Ledbetter, D.H., and Caskey, C.T. 1989. Alu polymerase chain reaction: A method for rapid isolation of human‐specific sequences from complex DNA sources. Proc. Natl. Acad. Sci. U.S.A. 86:6686‐6690.
  Describes Alu‐PCR of human DNA in complex sources.
   Ross et al., 1992. See above.
  Describes procedure for hybridization to YAC colony blots.
Internet Resources
   http://genome.wustl.edu/gsc
  Genome Sequencing Center homepage; includes overgo maker access.
   http://ftp.genome.washington.edu
  Source for RepeatMasker program.
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