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Signature-tagged mutagenesis: barcoding mutants for genome-wide screens

Nature Reviews Genetics volume 7, pages 929–939 (2006)Cite this article

Key Points

  • In signature-tagged mutagenesis (STM), molecular barcoding allows high-throughput screens by identifying mutants with reduced or increased adaptation to certain environments.

  • STM has undergone many technical adaptations and improvements since its invention in 1995. These include the use of pre-selected tags, different types of mutagen, and labelling techniques and high-density arrays for hybridization analysis. Related techniques such as TraSH, MATT and DeADMAn use DNA sequences that flank transposon insertions as signature tags.

  • The majority of STM studies have been carried out on pathogenic bacteria, and these have resulted in a wealth of insights into a broad range of virulence processes. Screens have also been carried out to identify host- and tissue-specific virulence factors, and with immune-deficient mouse strains to uncover genes encoding proteins that protect the pathogen against specific host immune mechanisms.

  • Signature tags were incorporated into genomes of strains in the Saccharomyces cerevisiae deletion mutant collection. The collection has been subjected to numerous screens, which have identified thousands of genes that are involved in diverse metabolic processes. The yeast deletion mutant library can also be screened for other characteristics such as haploinsufficiency, chemical sensitivity and synthetic lethality.

  • Recently, signature tagging has been used in conjunction with small interfering RNA screens in diploid mammalian cells. This approach has already led to the discovery of a novel tumour suppressor, and offers considerable potential for large-scale analysis of mammalian gene function.

Abstract

DNA signature tags (molecular barcodes) facilitate functional screens by identifying mutants in mixed populations that have a reduced or increased adaptation to a particular environment. Many innovative adaptations and refinements in the technology have been described since its original use with Salmonella; they have yielded a wealth of information on a broad range of biological processes — mainly in bacteria, but also in yeast and other fungi, viruses, parasites and, most recently, in mammalian cells. By combining whole-genome microarrays and comprehensive ordered libraries of mutants, high-throughput functional screens can now be achieved on a genomic scale.

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Figure 1: Original signature-tagged mutagenesis of Salmonella.
Figure 2: Methods for generating pools of tagged mutants.
Figure 3: Methods for the detection of signature tags.
Figure 4: Differential STM screen.
Figure 5: RNAi screen with barcoding.

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Acknowledgements

D.W.H. is funded by grants from the UK Medical Research Council and the Wellcome Trust. C.B. is funded by the Canadian Institute of Health Research, Genome Canada and Genome Ontario.

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Authors and Affiliations

  1. Department of Infectious Diseases, Centre for Molecular Microbiology and Infection, Imperial College London, Armstrong Road, London, SW7 2AZ, UK

    Piotr Mazurkiewicz, Christoph M. Tang & David W. Holden

  2. Banting and Best Department of Medical Research, University of Toronto, 112 College Street, Toronto, M5G 1L6, Ontario, Canada

    Charles Boone

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FURTHER INFORMATION

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Glossary

Genetic footprinting

A technique in which a large population of cells is subjected to transposon mutagenesis, then divided and subjected to different selective pressures. PCR on individual genes determines the presence or absence of corresponding mutant strains after selection.

Virulence

The degree of pathogenicity, or ability to cause a disease.

Pathogenicity island

A distinct region of the genome, usually with an altered G and C content and containing one or more virulence genes, that was probably acquired by horizontal gene transfer.

Cell envelope

The cell membrane and outer membrane, if one is present, together with the cell wall; Mycobacteria have a very complex cell wall that is rich in glycolipids, especially mycolic acids.

Innate immunity

'General' or 'non-specific' immune responses; the first line of defence against a pathogen.

Adhesins

Molecules that mediate the attachment of an organism to a surface.

Latency

The persistence of a pathogen in the host cell without necessarily causing signs of disease.

Zoonotic

A pathogen that primarily affects animals but is transmissible to humans.

Apicomplexan

A type of protozoan parasite that uses a unique form of actin-based motility and has a complex life cycle.

Small interfering RNAs

Small antisense RNAs (20–25 nucleotides) that are generated from specific dsRNAs that trigger RNAi. They serve as guides for the cleavage of homologous mRNA in the RNA-induced silencing complex.

Short hairpin RNAs

Small RNAs that form hairpins that can induce sequence-specific silencing in mammalian cells through RNAi, when produced exogenously and transfected into the cell or when expressed endogenously.

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Mazurkiewicz, P., Tang, C., Boone, C. et al. Signature-tagged mutagenesis: barcoding mutants for genome-wide screens. Nat Rev Genet 7, 929–939 (2006). https://doi.org/10.1038/nrg1984

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