Oligonucleotide Microarrays for Clinical Diagnosis of Copy Number Variation and Zygosity Status

David T. Miller1, Yiping Shen1, Bai‐Lin Wu1

1 Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 8.12
DOI:  10.1002/0471142905.hg0812s74
Online Posting Date:  July, 2012
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Detection of submicroscopic genomic copy number variation is now considered the first‐tier clinical test—in place of standard G‐banded karyotyping—in the evaluation of children with unexplained developmental delay, intellectual disability, autism spectrum disorders, or congenital anomalies. Fluorescence in situ hybridization (FISH) was the first molecular method for detection of submicroscopic genomic copy number variants (CNVs), but microarray‐based comparative genomic hybridization (array CGH) has a much higher diagnostic yield for these patients when compared to traditional cytogenetic methods such as karyotype and FISH. This unit focuses on oligonucleotide arrays, including updated information about detection of long contiguous stretches of homozygosity (LCSH) through inclusion of single‐nucleotide polymorphism (SNP) probes. Most clinical laboratories now offer arrays with some level of probe coverage throughout the genome, and many are offering detection of LCSH. Updated guidelines for array design and result interpretation are reviewed. Curr. Protoc. Hum. Genet. 74:8.12.1‐8.12.17. © 2012 by John Wiley & Sons, Inc.

Keywords: copy number variant; CNV; genomic imbalance; molecular diagnostics; array comparative genomic hybridization; CGH; aCGH

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Table of Contents

  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • 100 to 150 ng/µl genomic DNA in TE buffer:
    • Sample (test) DNA (isolated from blood, see unit 14.4)
    • Control (reference) DNA (e.g., normal male or female):
    • NA18507 (Yoruban Male)
    • NA18517 (Yoruban Female)
    • NA12891 (European Male)
    • NA12878 (European Female)
    • NA18579 (Chinese Female)
  • Nuclease‐free water
  • 10× reaction buffer C (provided with Rsa I; Promega)
  • 10 U/µl Alu I (Promega)
  • 10 U/µl Rsa I (Promega)
  • QIAprep Spin Miniprep Kit (Qiagen; cat. nos. 27104, 27106) with Buffers PB, PE, and EB
  • Ethanol (denatured; Fisher)
  • BioPrime Array CGH Genomic Labeling System (Invitrogen; cat. no. 18095011), with:
    • 2.5× Random primers solution (octamers)
    • 40 U/µl exo‐Klenow fragment
    • Stop buffer
  • 10× dUTP (nucleotide mix; PerkinElmer)
  • 2′‐Deoxyuridine conjugated with cyanine 3 and cyanine 5 (Cy3‐dUTP and Cy5‐dUTP; PerkinElmer)
  • 1× TE buffer, pH 8.0 (Promega)
  • Oligonucleotide aCGH Hybridization kit, large volume (Agilent, 5188‐5380), containing:
    • 10× Blocking Agent
    • 2× Hybridization buffer
  • Cot‐1 DNA (Invitrogen)
  • Oligonucleotide aCGH wash buffers 1 and 2 (Agilent)
  • Spectrophotometer (e.g., NanoDrop ND‐1000, Thermo Fisher Scientific)
  • Thermal cycler
  • Centrifuge
  • 1.5‐ml tubes
  • Speedvac Concentrator
  • 37°C incubator
  • Microcon YM‐30 Centrifugal Filter Kit (Millipore; cat. no. 42410)
  • Microcentrifuge
  • Oligonucleotide microarray, e.g., Human Genome 244 k array (Agilent Technologies, G4411B)
  • Hybridization Oven (Agilent Technologies)
  • Coplin jar
  • Ozone detector (e.g., Teledyne Model 400E), optional
  • Ozone converter (e.g., Ozone Solutions “Ozone Interceptor”), optional
  • Array scanner (e.g., Agilent G2565AA or 2565 BA)
  • Computer workstation (e.g., Dell Optiplex GX745)
  • Feature Extraction software (included with purchase of array scanner)
  • CGH Analytics software (Agilent)
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Literature Cited

Literature Cited
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