Direct genomic selection (original) (raw)

• Protocol
• Published: January 2005
• Rose Veile1,
• Cynthia Helms1,
• Elaine R Mardis1,
• Anne M Bowcock1 &
• …
• Michael Lovett1

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

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

• Restriction enzymes _Sau_3AI (Promega), _Hin_fI (Invitrogen) and _Eco_RI (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

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

References

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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
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2. Rose Veile
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3. Cynthia Helms
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4. Elaine R Mardis
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5. Anne M Bowcock
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6. Michael Lovett
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Corresponding author

Correspondence toMichael Lovett.

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

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• Issue Date: January 2005
• DOI: https://doi.org/10.1038/nmeth0105-63

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