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Primer: genomic and proteomic tools for the molecular dissection of disease

Nature Clinical Practice Rheumatology volume 3, pages 580–589 (2007)Cite this article

Abstract

Completion of the Human Genome Project has been rapidly followed by the emergence of high-throughput technologies that combine automation, miniaturization, and many other strategies and tools to enable systematic surveys of genome composition and gene expression. Of particular relevance to the prevention and management of disease are technologies such as high-throughput DNA genotyping, microarray-based gene-expression profiling, and mass spectrometry-facilitated protein profiling—platforms that collectively support the comprehensive analysis of DNA sequence variants across the genome and the global gene and protein expression changes that distinguish health from disease. Now used extensively in all facets of biomedical investigation, genomic and proteomic tools are already beginning to pinpoint molecular variants that influence risk and outcome in common diseases, and to thereby inform and direct development of novel molecular biomarkers and drug targets. As evidenced by recent advances in DNA sequencing methods, continued improvements in the scope, power, and cost efficiency of genomic and proteomic technologies should ensure their capacity to provide the scale and depth of knowledge required for translating genome sequence information into major medical impact.

Key Points

  • Genomic sciences encapsulate a group of high-throughput technologies that enable the simultaneous systematic analysis of whole genomes or proteomes

  • Genomics research has identified single nucleotide polymorphisms as the major source of genome variation and provided powerful technology platforms to facilitate high-throughput scoring of these variants and their use in genome-wide association analysis

  • Microarrays are used in many facets of genomics research, enabling, for example, the simultaneous interrogation of hundreds of thousands of single nucleotide polymorphisms or expressed transcripts to be carried out

  • Advances in mass spectrometry instrumentation, automation and analytic methods have made mass spectrometry one of the most powerful platforms for high-throughput protein analysis

  • Continued improvements in genomic and proteomic technologies will increase their power, efficiency and, ultimately, clinical impact

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Figure 1: Strategies for disease gene mapping.
Figure 2: Two ultra-high-throughput single nucleotide polymorphism genotyping platforms for use in genome-wide association analyses.
Figure 3: Complementary DNA spotting and oligonucleotide microarray methods for comparative transcript profiling.

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Acknowledgements

This work was supported by grants from the Ontario Research Development and Challenge Fund and the Canadian Institutes for Health Research. E Walker is supported by a McLaughlin Centre for Molecular Medicine (MCMM) fellowship award and K Siminovitch is an MCMM Scientist and holder of a Canada Research Chair.

Author information

Authors and Affiliations

  1. where KA Siminovitch is a Senior Scientist. KA Siminovitch is also a Professor in the Departments of Medicine, EJ Walker is a Postdoctoral Fellow at the Mount Sinai Hospital, Samuel Lunenfeld and the Toronto General Research Institutes, Immunology and Medical Genetics and Microbiology at the University of Toronto, Toronto, Canada.,

    Erin J Walker & Katherine A Siminovitch

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  1. Erin J Walker
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Corresponding author

Correspondence to Katherine A Siminovitch.

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Supplementary information

Supplementary Figures 1

Genotyping single nucleotide polymorphisms by MALDI mass spectrometry. DNA templates for mass spectrometric analysis are prepared from genomic DNA from test samples using PCR to amplify short DNA fragments (~100 bp) that contain the target sequence. A primer annealing immediately upstream of the SNP on the target sequence is used with terminator deoxynucleotides and DNA polymerase to drive a primer extension reaction that creates amplified product representing one or the other of the SNP alleles. The amplified extension products are spotted onto chips and subjected to MALDI-TOF mass spectrometry. The allele-specific products differ in mass and generate distinct mass spectra that are interpreted using allele-calling software to assign SNP genotype. Abbreviations: MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PCR, polymerase chain reaction; SNP, single-nucleotide polymorphism. (JPG 375 kb)

Supplementary Figures 2

Schematic illustration of an ultra high-throughput sequencing strategy (454 Pyrosequencing). (A) Sequencing strategy. DNA templates are first fragmented and the fragments clonally amplified on beads within a microreactor formed by an emulsion of oil and water. (B) Sequencing reactions are performed in a picotitre plate, such that each plate carries about 1.6 million beads. dNTPs are flowed individually and sequentially across the plate in a fixed order. (C) Pyrosequencing chemistry. Incorporation of each dNTP results in the release of a pyrophosphate molecule. The pyrophosphate and adenosine 5’ phosphosulfate molecules are then converted by the sulfurylase enzyme to ATP, which provides energy for luciferase activation and light emission in parallel with single base addition. Abbreviations: ATP, adenosine triphosphate; dNTP, deoxynucleotide triphosphate; PPi, pyrophosphate. (PDF 599 kb)

Supplementary Figures 3

Mass spectrometry-mediated expression profiling. Samples are collected from affected individuals and controls and the proteins extracted, digested into peptide fragments, and then analyzed using two-dimensional high-performance liquid chromatography to separate the peptides according to charge and size. The separated peptides are directly subjected to mass spectrometric analysis using electrospray ionization or MALDI and tandem (MS/MS) mass spectrometry. Mass spectra are generated and database search programs used to identify the peptide components and proteins in each sample and the protein expression patterns that differ between disease and healthy states. Abbreviation: MALDI, matrix-assisted laser desorption/ionization. (JPG 96 kb)

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Walker, E., Siminovitch, K. Primer: genomic and proteomic tools for the molecular dissection of disease. Nat Rev Rheumatol 3, 580–589 (2007). https://doi.org/10.1038/ncprheum0595

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