Beads are good for you

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 878923-8927 
• 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. 8684-92
• 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 881143-7.

3
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
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.282142-6.
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
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Luminex beads are analyzed by flow cytometry
one well100 different beads100 reactions
Luminex 100
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Luminex, SBCE
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Luminex, critical factors
ddA
ddG
13
Illumina beads are analyzed through
fiberoptics-ducted fluorescence excitation
96-well plate -gt 0.5M beads
1 bundle of 50k fibers -gt 50k beads
1 fiber gt 1 bead
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Illumina genotyping Golden gate
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Illumina genotypes
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Reviewing the product-bead relationship
home
AGTT
snp1
snp1
snp2
CAGG
snp2
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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

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Multiplexing II advanced

• Run 100 genotyping rx in a single tube
• Sort each to the assigned sphere color by a zip
  code

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Multiplexing III, more advanced

• oops, the scheme below does not work because
  different fluors will label the same ball.

20
Factoids
Luminex Illumina
Sphere types 100 1,500
Cost per snp 0.3 0.03 to 0.4
Genotypes/8 hrs 10K ?
Mx Th. Gen/day 120K 300K
templ. amplif. locus PCR whole genome
multiplex 12-50 50-1000