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Beads are good for you

Beads are good for you. and for genotyping Fan et al. 2005. Biotechniques 39, 583 (Illumina) Chen et al. 2000. Genome Res 10, 549 (Luminex). SNP Detection. Oligonucleotide ligation

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Beads are good for you

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  1. Beads are good for you and for genotyping Fan et al. 2005. Biotechniques 39, 583 (Illumina) Chen et al. 2000. Genome Res 10, 549 (Luminex)

  2. SNP Detection • Oligonucleotide ligation • AM Alves and FJ Carr. 1988. Dot blot detection of point mutations with adjacently hybridising synthetic oligonucleotide probes. Nucl. Acids Res. 16: 8733 • NICKERSON DA, R KAISER, S LAPPIN, J STEWART, LEROY HOOD, AND ULF LANDEGREN 1990 Automated DNA diagnostics using an ELISA-based oligonucleotide ligation assay PNAS 87:8923-8927  • Primer extension • Syvanen AC, Aalto-Setala K, Harju L, Kontula K, Soderlund H. 1990 A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics. 8:684-92 • Kuppuswamy MN, Hoffmann JW, Kasper CK, Spitzer SG, Groce SL, Bajaj SP. 1991 Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc Natl Acad Sci U S A. Feb 88:1143-7.

  3. DNA Ligase T TCGAAACTCATATC CAGCGGGAGTTAGAC …ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA… DNA Ligase Oligonucleotide Ligation 3’ 5’ TCGAAACTCATATCT CAGCGGGAGTTAGAC …ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA…

  4. Oligonucleotide Ligation Assay (OLA) amplify target by PCR ligate oligos detect ligation product

  5. Primer extension 3’ C TCGAAACTCATATCT C …ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA… DNA Pol C TCGAAACTCATATC …ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA…

  6. SNP1 C SNP2 C C SNP3 C SNP4 One tube = one reaction = one assay?

  7. Bead types rosary beads “test tube” bead = 5 uM 1 cM

  8. Beads can be marked Luminex beads Illumina beads 5.6 um, polystyrene 3 um, silica 1500 color-coded types 100 color-coded types Lee M, Walt DR. 2000 Anal Biochem.282:142-6. R. J. Fulton et al., Clin. Chem. 443, 1749 (1997)

  9. Beads can be derivatized Each bead type can be coated with one specific probe type: e.g. oligonucleotide =aggctcgatc

  10. Luminex beads are analyzed by flow cytometry one well=100 different beads=100 reactions Luminex 100

  11. Luminex, SBCE

  12. Luminex, critical factors ddA ddG

  13. Illumina beads are analyzed through fiberoptics-ducted fluorescence excitation 96-well plate -> 0.5M beads 1 bundle of 50k fibers -> 50k beads 1 fiber > 1 bead

  14. Illumina genotyping: Golden gate

  15. Illumina genotypes

  16. address TCAA TCAA GTCC GTCC Reviewing the product-bead relationship home AGTT snp1 snp1 snp2 CAGG snp2

  17. Multiplexing I: basic options • 100 sphere types • run 100 rx in 100 tubes • pool them and load them as a single well • not impressive, we do something like that now with fluorescent primers and the ABI 3100

  18. Multiplexing II: advanced • Run 100 genotyping rx in a single tube • Sort each to the assigned sphere color by a zip code

  19. Multiplexing III, more advanced • oops, the scheme below does not work because different fluors will label the same ball…….

  20. Factoids

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