Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Direct genomic selection

Nature Methods volume 2, pages 63–69 (2005)Cite this article

  • Genomic DNA, pooled isolates from individual patients or tissue samples

  • Restriction enzymes Sau3AI (Promega), HinfI (Invitrogen) and EcoRI (Invitrogen)

  • DNA polymerases: T4 DNA polymerase (Invitrogen), PfuUltra HF DNA polymerase (Stratagene)

  • 5× T4 DNA polymerase reaction mix: 5× T4 DNA polymerase buffer, 1 mM each dNTP

  • Oligonucleotide 3: 5′-CTCGAGAATTCTGGATCCTC-3′, Oligonucleotide 4: 5′-GAGGATCCAGAATTCTCGAGTT-3′

  • 10× annealing buffer: 100 mM Tris-HCl (pH 7.5), 1 M NaCl, 10 mM EDTA

  • T4 DNA ligase (Roche)

  • QIAquick PCR purification kit (Qiagen)

  • Purified BAC DNA, from clone encoding region of interest

  • Biotin-16-dUTP (Enzo Biochemicals)

  • 10× nick translation buffer: 500 mM Tris-HCl (pH 7.5), 100 mM MgCl2, 50 mM DTT, 400 μM each dNTP

  • [α-32P]dCTP (3,000 Ci/mmol) (Amersham)

  • DNA polymerase/DNase I (Roche)

  • Cot-1 DNA (Invitrogen)

  • 2× hybridization buffer: 1.5 M NaCl, 40 mM sodium phosphate buffer (pH 7.2), 10 mM EDTA (pH 8), 10× Denhardt's, 0.2% SDS

  • Streptavidin-coated paramagnetic beads and magnetic bead separator (Dynabeads M-280 Streptavidin)(Dynal)

  • Streptavidin bead binding buffer: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8) and 1 M NaCl

  • dNTP solution (25 mM each) (Invitrogen)

  • p-AMP1 plasmid DNA (Invitrogen)

  • DH10B electrocompetent Escherichia coli (Invitrogen)

Genomic DNA, pooled isolates from individual patients or tissue samples

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A flow diagram of the direct genomic selection process.

References

  1. Helms, C. et al. A putative RUNX1 binding site variant between SLC9A3R1 and RAT9 is associated with susceptibility to psoriasis. Nat. Genet. 35, 349–356 (2003).

    Article  CAS  Google Scholar 

  2. Pajukanta, P. et al. Familial combined hyperlipidemia is associated with upstream transcription factor 1 (USF1). Nat. Genet. 36, 371–376 (2004).

    Article  CAS  Google Scholar 

  3. Balmain, A., Gray, J. & Ponder, B. The genetics and genomics of cancer. Nat. Genet. (Suppl.) 33, 238–244 (2003).

    Article  CAS  Google Scholar 

  4. Kile, B.T. et al. Functional genetic analysis of mouse chromosome 11. Nature 425, 81–86 (2003).

    Article  CAS  Google Scholar 

  5. Lovett, M., Kere, J. & Hinton, L.M. Direct selection: a method for the isolation of cDNAs encoded by extended genomic regions. Proc. Natl. Acad. Sci. USA 88, 9628–9632 (1991).

    Article  CAS  Google Scholar 

  6. Lovett, M. Direct selection of cDNAs with large genomic DNA clones. In Molecular Cloning: A Laboratory Manual Edn. 3 Vol. 2 (eds. Sambrook., J. & Russell, D.W.) 11.98–11.133 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001).

    Google Scholar 

  7. Hacia, J.G. Resequencing and mutational analysis using oligonucleotide microarrays. Nat. Genet. 21, 42–49 (1999)

    Article  CAS  Google Scholar 

  8. Faham, M., Baharloo, S., Tomitaka, S., DeYoung, J. & Freimer, N.B. Mismatch repair detection (MRD): high-throughput scanning for DNA variations. Hum. Mol. Genet. 10, 1657–1664 (2001).

    Article  CAS  Google Scholar 

  9. Stengard, J.H. et al. Contributions of 18 additional DNA sequence variations in the gene encoding apolipoprotein E to explaining variation in quantitative measures of lipid metabolism. Am. J. Hum. Genet. 71, 501–517 (2002).

    Article  CAS  Google Scholar 

  10. Crawford, D.C. et al. Haplotype diversity across 100 candidate genes for inflammation, lipid metabolism, and blood pressure regulation in two populations. Am. J. Hum. Genet. 74, 610–622 (2004).

    Article  CAS  Google Scholar 

  11. Fullerton, S.M. et al. The effects of scale: variation in the APOA1/C3/A4/A5 gene cluster. Hum. Genet. 115, 36–56 (2004).

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Genetics, Washington University School of Medicine, Box 8232, 4566 Scott Avenue, St. Louis, 63110, Missouri, USA

    Stavros Bashiardes, Rose Veile, Cynthia Helms, Elaine R Mardis, Anne M Bowcock & Michael Lovett

Authors
  1. Stavros Bashiardes
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rose Veile
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Cynthia Helms
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Elaine R Mardis
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Anne M Bowcock
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Lovett
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Michael Lovett.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bashiardes, S., Veile, R., Helms, C. et al. Direct genomic selection. Nat Methods 2, 63–69 (2005). https://doi.org/10.1038/nmeth0105-63

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth0105-63

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing