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. Oligonucleotide Ligation …ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA… TCGAAACTCATATCTCAGCGGGAGTTAGAC DNA Ligase 3’ 5’ … ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA … TCGAAACTCATATCCAGCGGGAGTTAGAC T DNA Ligase

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

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

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

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

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

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 ddAddG

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

14. Illumina genotyping: Golden gate

15. Illumina genotypes

16. Reviewing the product-bead relationship AGTT CAGG home TCAA GTCC address TCAA GTCC snp1 snp2 snp1 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 LuminexIllumina Sphere types1001,500 Cost per snp0.30.03 to 0.4 Genotypes/8 hrs10K? Mx Th. Gen/day120K300K templ. amplif.locus PCRwhole genome multiplex12-5050-1000