Selection of a Platform for Mutation Detection

Victoria A. Joshi1,2,3, Debora Mancini‐DiNardo1,3, Birgit H. Funke1,2,3

1 Harvard Medical School‐Partners Healthcare Center for Genetics and Genomics, Cambridge, Massachusetts, 2 Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts, 3 null, null, null
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.15
DOI:  10.1002/0471142905.hg0715s56
Online Posting Date:  January, 2008
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Abstract

New mutation detection technologies must keep pace by becoming more cost-effective while offering improved technical sensitivity and higher throughput capacity. In recent years, the number of mutation detection platforms available to the clinical researcher has grown to a point where it is difficult to keep track of all available options as well as their benefits and pitfalls. This unit provides an entry point for a variety of researchers who wish to analyze samples for known or novel mutations and need to determine which platform is most suited for their particular needs. A practical guide is provided in this unit, including a brief overview, information on assay parameters, design and cost considerations, as well as platform flexibility and scalability of the assay. Although the focus here is on applications involving human disease, many of these platforms can be easily adapted to the study of other organisms. Curr. Protoc. Hum. Genet. 56:7.15.1-7.15.30. © 2008 by John Wiley & Sons, Inc.

Keywords: mutation detection; mutation scanning; genotyping

     
 
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Table of Contents

  • Introduction
  • Methods for Direct Mutation Detection
  • Methods for Mutation Scanning
  • Detection of Constitutional Copy Number Variation
  • Methylation Detection
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •  FigureFigure 7.15.1 Top: The principle of an ARMS assay designed to detect a T/C polymorphism. A and B represent reaction 1 for the T allele. The allele-specific primer for the T allele binds and can be extended, while the primer specific for the C allele has two mismatches and cannot be extended. C and D show the converse and represent reaction 2 for the C allele. Bottom: The result for three hypothetical samples are shown. Two internal control primer pairs give rise to the top and bottom band in each reaction.
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  •  FigureFigure 7.15.2 (A) Genotyping results for an SNP present in the VKORC1 gene. Red circles represent patients with homozygous wild-type genotypes, green triangles represent patients with heterozygous genotypes, blue diamonds represent patients with homozygous mutant genotypes, and grey squares represent negative controls. The sample designated by the X symbol lies outside the acceptable range for genotype calls and was deemed invalid. Representative quantitative PCR results for a homozygous mutant sample (B), a homozygous wild-type sample (C), and a heterozygous sample (D).
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  •  FigureFigure 7.15.3 The principles of the original (hME; A) as well as the newer version (iPLEX; B) of the MassARRAY technology are shown for a hypothetical T/A polymorphism. hME reaction mixes are designed such that a dideoxy nucletotide is incorporated at the first position after the primer when allele 1 is present. For the other allele, the reaction extends for one additional base and terminates at position 2. iPLEX reaction mixes use mass modified nucleotides, which eliminates the need for an extension beyond the first (polymorphic) base.
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  •  FigureFigure 7.15.4 Plating scheme to genotype 32 multiplexes in 12 samples. Samples are first plated onto four 96-well masterplates in the order shown and then transferred to the 384-well plate to generate multiple identical sections of 12.
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  •  FigureFigure 7.15.5 Representative DNA sequence chromatograms, as viewed using Mutation Surveyor. The upper chromatogram (pair) in each image represents the forward strand and the lower chromatogram (pair) represents the reverse strand. (The uppermost and lowermost strands are the reference sequences.) (A) A homozygous missense mutation. (B) A heterozygous missense mutation. (C) Scrambled sequence due to a CTTCT deletion.
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  •  FigureFigure 7.15.6 The two left panels show MLPA results generated with the GeneMarker software (Softgenetics). In both cases, an apparent single probe deletion was present (symbolized by the red box). Subsequent sequence analysis of the probe binding sites revealed a heterozygous variant for one (top right) and a heterozygous deletion for the other sample (bottom right, deletion indicated by the brown bar).
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Literature Cited

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 Internet Resources
 Primer design
    http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

Web-based primer design tool (Primer 3).

    http://seq.yeastgenome.org/cgi-bin/web-primer

Web-based primer design tool (Saccharomyces Genome Database).

    http://ngrl.man.ac.uk/SNPCheck/

Tool for batch-checking SNPs in oligonucleotide primers (National Genetics Reference Laboratory, UK).

    http://www.ncbi.nlm.nih.gov/SNP/

dbSNP database at NCBI.

    http://snpper.chip.org/

Web-based application to look for known SNPs in public databases.

    http://www.ensembl.org/Homo_sapiens/index.html

Ensembl genome database.

    http://genome.ucsc.edu/

UCSC genome bioinformatics site.

 RFLP
    http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechCAPS.shtml

Information on the principle underlying the RFLP (CAPS) analysis.

    http://tools.neb.com/NEBcutter2/index.php

E-cutting tool (New England Biolabs).

    http://www.firstmarket.com/cutter/cut2.html

E-cutting tool (Webcutter 2.0).

    http://watcut.uwaterloo.ca/watcut/watcut/template.php

E-cutting tool (University of Waterloo, Canada).

    http://www.med.yale.edu/genetics/ward/tavi/p15.html

Gel electrophoresis, comparison of agarose type (non-polymorphic loci).

 Microarray-based ASO
    http://www.affymetrix.com/support/downloads/manuals/genomewidesnp6_manual.pdf

Genome-Wide Human SNP Array 6.0 User Manual (pdf file).

    http://www.affymetrix.com/products/arrays/specific/genome_wide_snp6/genome_wide_snp_6.affx

Affymetrix Website with description of Genome-Wide Human SNP Array 6.0.

    http://www.nanogen.com

Information on Nanogen microarray platforms.

    http://www.chem.agilent.com/Scripts/PCol.asp?lPage=494

Information on Agilent microarray platforms.

    http://www.asperbio.com/

Information on Asper microarray platforms.

 Luminex xMAP-based ASO
    http://www.tmbioscience.com/productsmain.php

Luminex Tag-It mutation detection kits.

 Universal bead-based ASO
    http://www.illumina.com/pages.ilmn=ID=37

Illumina DNA analysis solutions.

 TaqMan
    http://docs.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_042379.pdf.

ABI custom TaqMan design information (pdf file).

 Invader
    http://www.universalinvader.com/twt/uic/index.vm

Third Wave Technologies Website describing design process for Universal Invader.

 MassARRAY
    http://www.sequenom.com/Assets/pdfs/appnotes/8876-006.pdf

Sequenom application for the iPLEX assay.

    http://snpper.chip.org/

Web-based application maintained by Children's Hospital Boston to look for known in public databases.

 Heteroduplex analysis (dHPLC)
    http://www.transgenomic.com/

Transgenomic, a major provider of dHPLC separation matrix and instrumentation.

 DNA sequencing
    http://www.affymetrix.com/products/application/sequence_analysis.affx

Affymetrix Website describing GeneChip CustomSeq Resequencing Arrays.

 MLPA
    http://www.mlpa.com

Webpage of MRC Holland, a company that sells a large number of MLPA kits.

    http://www.mrc-holland.com/pages/products_methylation_specificpag.html

Information on MLPA-based methylation detection kits sold by MRC Holland.

    http://www.mlpa.com/pages/support_desing_synthetic_probespag.html

Instructions on the design of synthetic MLPA probes.

    http://www.mlpa.com/pages/support_mlpa_analysis_overigpag.html

List of available analysis tools for MLPA data analysis.

 Methylation-specific PCR
    http://www.chemicon.com/home.asp

Chemicon (Millipore), a supplier for commercial MSP kits.

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