Detecting APC Gene Mutations in Familial Adenomatous Polyposis (FAP)

Babi Ramesh Reddy Nallamilli1, Madhuri Hegde1

1 Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 10.8
DOI:  10.1002/cphg.29
Online Posting Date:  January, 2017
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Hereditary forms of colorectal cancer (CRC) account for up to 5% of total cases. Familial adenomatous polyposis (FAP) is an autosomal dominant condition affecting nearly 1 in 5000 people and accounts for only about 1% of all CRCs. It is characterized by the progressive development of hundreds to thousands of adenomatous colon polyps. The gene associated with FAP (APC) contains 15 coding exons. The mutation spectrum of the APC gene is broad in that 87% of causative mutations are point mutations (including other sequence variants) and around 10% to 15% are intragenic deletions and duplications. The strategy for molecular diagnostic testing for FAP involves initial full sequence analysis of APC for sequence variants followed by screening for deletion/duplications using microarray‐based comparative genomic hybridization (array CGH) or Multiplex Ligation‐dependent Probe Amplification (MLPA). Recently, next generation sequencing (NGS)‐based targeted gene analysis has become clinically available for detection of point mutations and other sequence variants. This unit discusses detailed protocols for an NGS‐based sequencing assay, PCR‐based Sanger sequencing, and array CGH. © 2017 by John Wiley & Sons, Inc.

Keywords: APC gene; NGS‐based sequencing; Sanger sequencing; array CGH

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Table of Contents

  • Introduction
  • Basic Protocol 1: Targeted Next Generation Sequencing of APC Using IDT xGen Lockdown Probes
  • Support Protocol 1: DNA Isolation from Whole Blood
  • Basic Protocol 2: Sanger Sequencing of Entire APC Coding Region to Detect Point Mutations
  • Basic Protocol 3: Microarray‐Based Comparative Genomic Hybridization to Detect Deletions and Duplications in APC Gene
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Targeted Next Generation Sequencing of APC Using IDT xGen Lockdown Probes

  • 5 µM APC exon‐specific forward primer
  • 5 µM APC exon‐specific reverse primer
  • Genomic DNA from FAP patient
  • 10× PCR buffer with MgCl 2 (e.g., Roche)
  • 10 mM dNTPs (e.g., Roche)
  • 5 U/µl FastStart Taq DNA polymerase (e.g., Roche)
  • Nuclease‐free water
  • 2% agarose gel
  • BigDye Terminator v3.1 Cycle Sequencing Kit (e.g., Thermo Fisher Scientific) containing:
    • 5× sequencing buffer
    • BigDye Terminator v3.1 ready reaction mix
    • M13 forward or M13 reverse primer
  • Melatonin (e.g., Sigma, cat. no. M5250‐1G)
  • NanoDrop spectrophotometer
  • 96‐well PCR plate
  • Centrifuge with plate spinning adaptor
  • Thermal cycler
  • MultiScreen PCR purification plate (e.g., Millipore)
  • MultiScreen vacuum manifold (e.g., Millipore)
  • PCR plate mixer
  • 96‐well Performa DTR Version 3 purification plates (e.g., Egde BioSystems)
  • Septa for 96‐well plate
  • Vortex
  • ABI 3730 sequencer
  • APC reference file (available at NCBI Web site)
  • Mutation Surveyor software (e.g., SoftGenetics)
  • Additional reagents and equipment for agarose gel electrophoresis (Voytas, )
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Literature Cited

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