The BAC Resource: Tools for Array CGH and FISH

Norma J. Nowak1, Antoine M. Snijders2, Jeffrey M. Conroy1, Donna G. Albertson2

1 Roswell Park Cancer Institute and SUNY at Buffalo, Buffalo, New York, 2 University of California, San Francisco, San Francisco, California
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 4.13
DOI:  10.1002/0471142905.hg0413s46
Online Posting Date:  August, 2005
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Abstract

Bacterial Artificial Chromosome (BAC) vector clones carrying human DNA were chosen as the intermediate templates for the sequencing of the human genome due to their inherent stability and fidelity to the genome sequence from which they were derived. In this unit, we describe a set of protocols for BAC‐based array comparative genomic hybridization (aCGH) that comprise the generation of targets for printing solutions onto glass slides, the subsequent hybridization steps, and CGH analysis of a test sample compared to a reference normal sample. The BAC clones through their sequence allow the extent and gene content of numerical aberrations to be delineated by aCGH, and also provide cytogeneticists with tools for subsequent validation or fine mapping studies.

Keywords: Comparative Genomic Hybridization (CGH); Fluorescence In Situ Hybridization (FISH); Bacterial Artificial Chromosomes (BAC); microarrays; Copy Number Aberrations (CNA); Genomics; RPCI‐11

     
 
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Table of Contents

  • Strategic Planning
  • Precautions
  • Basic Protocol 1: BAC Selection and DNA Preparation for BAC CGH Arrays
  • Support Protocol 1: Glycerol Stock Archives and Replicas for Generating BAC CGH Arrays
  • Support Protocol 2: Assay for T1 Phage Contamination
  • Basic Protocol 2: Generation of LM‐PCR Product BAC Printing Solutions
  • Support Protocol 3: PicoGreen dsDNA Quantitation for BAC LM‐PCR Products
  • Basic Protocol 3: Printing BAC Arrays on Glass Slides
  • Basic Protocol 4: Random Primed Labeling and aCGH of Genomic DNA to BAC Arrays
  • Support Protocol 4: DNA Preparation of Test and Control Samples for aCGH Analysis
  • Support Protocol 5: WGA DNA Amplification for Low‐yield DNA Samples
  • Basic Protocol 5: Image and Data Analysis
  • Support Protocol 6: Verification of Copy Number Aberrations (CNAs)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: BAC Selection and DNA Preparation for BAC CGH Arrays

  Materials
  • LB agarose plates with chloramphenicol (see recipe), 7 × 7 grid, 22 × 22–cm
  • BAC clones (RPCI‐11 human) supplied by RPCI Microarray and Genomics Facility (see ) as single‐colony glycerol stock cultures in 96‐ or 384‐well plates (store at −80°C)
  • LB broth with chloramphenicol (see recipe)
  • 70% ethanol
  • 10 mM Tris·Cl, pH 8.5 ( appendix 2D)
  • Sterile toothpicks (CPMB UNIT )
  • 48‐well growth blocks (Qiagen cat. no. 19577)
  • Repeat pipettor (Eppendorf 22‐26006) with 50‐ml Combitip
  • Airpore strips (Qiagen cat. no. 19571)
  • Shaking incubator (New Brunswick C25), equipped with microtiter plate holder (New Brunswick, cat no. TTR‐221)
  • R.E.A.L. Prep 96 BioRobot Kit (Qiagen 26171)
  • Refrigerated centrifuge capable of 6000 × g, with microtiter plate carrier
  • BioRobot 3000 (Qiagen)
  • Additional reagents and equipment for PicoGreen quantitation of DNA (see protocol 5)

Support Protocol 1: Glycerol Stock Archives and Replicas for Generating BAC CGH Arrays

  Materials
  • LB broth containing glycerol and chloramphenicol (see recipe)
  • BAC clones (RPCI‐11 human) supplied by RPCI Microarray and Genomics Facility (see ) as single‐colony glycerol stock cultures in 96‐ or 384‐well plates (store at −80°C)
  • 96‐well, deep‐block sterile plates (Costar cat no. 3961)
  • Cryolabels (Marsh)
  • Long 250‐µl pipet tips
  • Airpore strips (Qiagen 19571)
  • Shaking incubator (New Brunswick C25), equipped with microtiter plate holder (New Brunswick, cat no. TTR‐221)
  • 96‐pin replication tool (V&P Scientific)
  • Cryoracks for −80°C freezer
  • Additional reagents and equipment for assaying BAC clones for T1 phage contamination (see protocol 3)

Support Protocol 2: Assay for T1 Phage Contamination

  Materials
  • E. coli strain DH10B (Invitrogen)
  • LB broth without chloramphenicol (see recipe; omit antibiotic)
  • LB top agarose (see recipe) in 500‐ml bottles
  • LB agarose plates without chloramphenicol (see recipe, omit antibiotic), 22 × 22–cm, nongridded (store up to 2 days inverted at 4°C, if not to be used immediately)
  • Shaking incubator (New Brunswick C25)
  • 42°C water bath

Basic Protocol 2: Generation of LM‐PCR Product BAC Printing Solutions

  Materials
  • 10× One‐Phor‐All Buffer Plus (Amersham)
  • Sterile UltraPure distilled H 2O, DNAse‐ and RNAse‐free (Invitrogen)
  • 10 U/µl MseI restriction enzyme (New England Biolabs; other similar enzymes can be used with compatible linkers)
  • 20 to 500 ng/µl BAC DNA (96 unique DNAs can be processed per plate; see protocol 1)
  • 100 µM Primers 1 and 2 (see recipe)
  • 10 mM ATP (Invitrogen)
  • 5 U/µl T4 DNA ligase (Invitrogen)
  • 10× Expand Long Template PCR buffer no. 1 (Roche)
  • 10 mM and 25 mM 4dNTP mix (see appendix 2D but prepare working solutions at 10 mM and 25 mM)
  • Expand Long Template DNA polymerase mix containing Taq and Pwo DNA polymerase, 3.5 U/µL (Roche)
  • 10× Platinum Taq PCR buffer (Invitrogen)
  • 25 mM MgCl 2
  • 5 U/µl Platinum Taq DNA polymerase (Invitrogen)
  • 1.5% agarose gel containing 0.5 µg/ml ethidium bromide (unit 2.7)
  • 100‐bp DNA ladder (Invitrogen)
  • 100% ethanol, prechilled to −20°C
  • 70% ethanol, ice‐cold
  • 20% (v/v) dimethylsulfoxide (DMSO; Fisher) in UltraPure distilled water
  • 0.2‐ml polypropylene PCR 12‐tube strips with separate 12‐cap strips (ABGene #AB‐1113), free of RNase, DNase, DNA, pyrogens, and PCR inhibitors.
  • Multichannel pipettors and aerosol‐barrier pipet tips
  • Thermal cycler capable of ramping at ∼1.3°C/min (PE GeneAmp 9600)
  • BioRobot 3000 (Qiagen)
  • 15‐ml tubes
  • 55‐ml reagent reservoirs for use with multichannel pipettor (Cole Parmer)
  • 96‐well thermal cycler plates (Costar cat. no. 6511)
  • 96‐well foil plate seals (Scotch 425, 3M)
  • 45°C hybridization oven (e.g., Hybaid)
  • Refrigerated centrifuge with microtiter plate carrier
  • 384‐well V‐bottom plates (Genetix)
  • Hydra‐PP workstation (Robbins Scientific)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Support Protocol 3: PicoGreen dsDNA Quantitation for BAC LM‐PCR Products

  Materials
  • PicoGreen dsDNA Quantitation Reagent and Kit (Molecular Probes) containing:
    • Component A: 200× PicoGreen dsDNA quantitation reagent (dilute to 1× with 1× TE buffer before use)
    • Component B: 20× TE buffer (dilute to 1× with nuclease‐free H 2O before use)
    • Component C: Lambda DNA standard (100 µg/ml)
  • 20% (v/v) dimethylsulfoxide (DMSO; Fisher) in UltraPure distilled water
  • LM‐PCR products (resuspended in 20 µl 20%DMSO; see protocol 4)
  • Black 96‐well flat‐bottom plates (Corning or Falcon)
  • Multichannel pipettors
  • SpectroMAX GeminiXS microtiter plate fluorescence reader (Molecular Devices)

Basic Protocol 3: Printing BAC Arrays on Glass Slides

  Materials
  • Sterile UltraPure distilled H 2O, DNAse‐ and RNAse‐free (Invitrogen)
  • 384‐well plate(s) containing printing solutions (see protocol 4)
  • Syto 61 staining solution (see recipe)
  • MicroGridII TAS arrayer (Genomic Solutions)
  • Microspot 10K pins (48 pins in 4 × 12 configuration; Genomic Solutions)
  • Bath sonicator (e.g., Health Sonics, http://www.healthson:cs.com
  • Nexterion Type A slides (Schott)
  • Antistatic brush
  • UV cross‐linker (e.g., Stratalinker from Stratagene)
  • Coplin jars
  • GenePix 4200A microarray scanner (Molecular Devices)
NOTE: Never place anything on the platform (but slides or plates in the proper holders).

Basic Protocol 4: Random Primed Labeling and aCGH of Genomic DNA to BAC Arrays

  Materials
  • Cot‐1 DNA, human (Invitrogen)
  • 3 M sodium acetate, pH 5.2 (purchase from Ambion; also see appendix 2D)
  • 100% ethanol, prechilled to −20°C
  • 70% ethanol, ice‐cold
  • 100 to 500 ng/µL test DNA sample ( protocol 8)
  • 100 to 500 ng/µL control DNA sample ( protocol 8)
  • BioPrime DNA Labeling System (Invitrogen) including:
    • 2.5× Random Primer solution
    • 40 U/µl exo‐Klenow fragment
  • 10× dNTP mix (see recipe)
  • 1 µM Cy3‐ labeled dCTP (PE Life Sciences cat. no. NEL576)
  • 1 µM Cy5‐labeled dCTP ( PE Life Sciences cat. no. NEL577)
  • TE buffer, pH 8.0 (optional; appendix 2D)
  • UltraPure H 2O (Invitrogen)
  • 100 µg/µl yeast tRNA (Invitrogen, cat. no. 15401‐029)
  • SlideHyb Buffer #3 (Ambion), prewarmed to 37°C
  • BAC arrays on glass slides (see protocol 6)
  • 20× SSC ( appendix 2D)
  • 20% SDS ( appendix 2D)
  • UV spectrophotometer
  • 95° to 100°C heating block
  • Spin columns (Qiagen, cat no. 28106)
  • Spectrolinker UV cross‐linker (Spectroline Products)
  • GeneTAC HybStation (Genomic Solutions)
  • Sorvall Legend RT centrifuge
  • GenePix 4200A microarray scanner (Molecular Devices)
  • Additional reagents and equipment for DNA quantitation ( appendix 3D)

Support Protocol 4: DNA Preparation of Test and Control Samples for aCGH Analysis

  Materials
  • Xylene (Fisher)
  • 70% and 100% ethanol
  • Puregene DNA purification kit (Gentra) containing:
    • Cell lysis solution
    • 20 mg/ml proteinase K
    • 4 mg/ml RNase
    • Protein precipitation solution
    • DNA hydration solution
  • Isopropanol
  • 20 mg/ml glycogen solution (optional; Gentra)
  • Kontes microcentrifuge tube pestle (Fisher)
  • 65°C water bath
  • Spectrofluorimeter (e.g., NanoDrop ND1000 from NanoDrop Technologies; http://www.nanodrop.com)
  • Additional reagents and equipment for spectrofluorimetric quantitation of DNA (APPENDIX appendix 3D)

Support Protocol 5: WGA DNA Amplification for Low‐yield DNA Samples

  Materials
  • High‐molecular‐weight (>40kb) genomic DNA sample
  • UltraPure H 2O (Invitrogen)
  • GenomiPhi DNA Amplification Kit (Amersham Biosciences) including:
    • Sample buffer
    • Reaction buffer
    • Enzyme mix
    • Control DNA
  • 500‐µl microcentrifuge tubes
  • 95° and 65°C heating blocks
  • Mini Quick Spin Columns (Roche cat no. 1814419)
  • 1.5‐ml microcentrifuge tubes, sterile
  • UV spectrophotometer
  • Additional reagents and equipment for DNA quantitation ( appendix 3D)

Basic Protocol 5: Image and Data Analysis

  Materials
  • Sample DNA
  • Marker‐specific primers
  • 5 U/µl Platinum Taq DNA polymerase (Invitrogen)
  • 10× PCR reaction buffer (Invitrogen)
  • 2.5% agarose gel (unit 2.7)
  • SYBR Green I (BioWhittaker)
  • 1× TAE buffer ( appendix 2D)
  • DIG‐Nick Translation Mix or Biotin‐Nick Translation Mix (Roche)
  • Cultured cells or interphase tumor nuclei as FISH targets (see unit 4.3)
  • Fluorescein‐conjugated anti‐digoxigenin Fab fragments or Avidin‐Rhodamine for probe detection (Roche)
  • Thin‐walled PCR tubes
  • Tetrad thermal cycler (MJ Research)
  • Typhoon 8600 imaging system (Amersham)
  • ImageQuant software (Amersham)
  • Fluorescence microscope (e.g., Nikon)
  • EasyFISH software (Applied Spectral Imaging)
  • Additional reagents and equipment for multiplex PCR (unit 9.3), agarose gel electrophoresis (unit 2.7), isolation of genomic DNA ( appendix 3B), and FISH (unit 4.3)
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Figures

Videos

Literature Cited

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Internet Resources
   http://microarrays.roswellpark.org/
  RPCI Microrray and Genomics Facility: Web site for RPCI BAC Resources and aCGH services
   http://www.ncbi.nlm.nih.gov/
  National Center for Biotechnology Information (NCBI): A national resource for molecular biology information.
   http://www.ncbi.nlm.nih.gov/genome/cyto/hbrc.shtml
  NCBI Human BAC Resource: This resource provides genome‐wide resource of large‐insert clones that will help integrate cytogenetic, radiation‐hybrid, linkage, and sequence maps of the human genome.
   http://genome.ucsc.edu/cgi‐bin/hgGateway?org=human
  UCSC Human Genome Browser Gateway: This site contains the reference sequence and working draft assemblies for the human genome.
   http://www.ensembl.org/Homo_sapiens/
  Ensembl Human Genome Browser: This site has an interactive software system that produces and maintains automatic annotation on the human genome.
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