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Designing and optimizing comparative anchor primers for comparative gene mapping and phylogenetic inference

Nature Protocols volume 2, pages 3022–3030 (2007)Cite this article

Abstract

Here we describe protocols for designing, optimizing and implementing conserved anchor primers for use in genome mapping or phylogenetic applications, with particular emphasis on homologous gene sequences among mammals. The increasing number of whole genome sequences in public databases makes this approach applicable across a wide range of taxa. Genome sequences from representatives of two or more divergent subclades within a taxonomic group of interest are used to identify candidate local alignments (i.e., exons, exons spanning introns or conserved 5′- or 3′-untranslated regions) that contain sequences with appropriate variability for the chosen downstream application. PCR primers are designed to maximize amplification success across a broad range of taxa, and are optimized under a touchdown thermocycling protocol. Based on the initial optimization results, primers are selected for application in a diverse sampling of species, or for mapping the genome of a target species of interest. We discuss factors that have to be considered for experimental design of broad-scope phylogenetic studies. With this protocol, primers can be designed, optimized and implemented within as little as 1–2 weeks.

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Figure 1
Figure 2: Example of an annotated DNA sequence used as input for Primer3.
Figure 3: PCR amplification results of three pairs of PCR primers (a, b and c), each targeting a different nuclear gene locus, tested in 12 mammals from different orders and a water-only negative (−) control.
Figure 4: PCR amplification results for a successful nuclear primer pair (shown in Fig. 3, primer c) tested in 47 different eutherian mammal DNAs, suitable for large-scale phylogenetic analysis.

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Acknowledgements

We thank Jan Janečka and Alison Wilkerson for helpful comments on this manuscript. This work was funded by the National Institutes of Health and the National Science Foundation.

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Authors and Affiliations

  1. Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, 77843-4458, Texas, USA

    William J Murphy

  2. Laboratory of Genomic Diversity, National Cancer Institute-Frederick, Frederick, 21702, Maryland, USA

    Stephen J O'Brien

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  1. William J Murphy
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  2. Stephen J O'Brien
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Correspondence to William J Murphy or Stephen J O'Brien.

Supplementary information

Supplementary Table 1

Conserved PCR primers optimized and applied in mammalian phylogenetic studies (PDF 32 kb)

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Murphy, W., O'Brien, S. Designing and optimizing comparative anchor primers for comparative gene mapping and phylogenetic inference. Nat Protoc 2, 3022–3030 (2007). https://doi.org/10.1038/nprot.2007.429

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