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
– 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.
Oligonucleotide Ligation
3’ 5’
TCGAAACTCATATCT CAGCGGGAGTTAGAC
…ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA…
DNA Ligase
T
TCGAAACTCATATC CAGCGGGAGTTAGAC
…ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA…
DNA Ligase
Oligonucleotide Ligation Assay (OLA)
amplify target by PCR
ligate oligos
detect
ligation
product
Primer extension
3’
TCGAAACTCATATCT C
C
…ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA…
DNA Pol
C
TCGAAACTCATATC
…ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA…
One tube = one reaction = one assay?
SNP1
C
SNP2
C
SNP3
SNP4
C
C
Bead types
rosary beads
“test tube” bead
=
5 uM
1 cM
Beads can be marked
Luminex beads
5.6 um, polystyrene
Illumina beads
3 um, silica
1500 colorcoded types
100 colorcoded types
R. J. Fulton et al., Clin. Chem. 443,
1749 (1997)
Lee M, Walt DR. 2000 Anal
Biochem.282:142-6.
Beads can be derivatized
Each bead type can be coated
with one specific probe type:
e.g. oligonucleotide
=aggctcgatc
Luminex beads are analyzed by flow cytometry
one well=100 different
beads=100 reactions
Luminex 100
Luminex, SBCE
Luminex, critical factors
ddA ddG
Illumina beads are analyzed through
fiberoptics-ducted fluorescence excitation
1 bundle of 50k
fibers -> 50k
beads
1 fiber > 1 bead
96-well
plate ->
0.5M
beads
Illumina genotyping:
Golden gate
Illumina genotypes
Reviewing the product-bead relationship
home
address
AGTT
TCAA
snp1
TCAA
GTCC
CAGG
GTCC
snp2
snp1
snp2
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
Multiplexing II: advanced
• Run 100 genotyping rx in a single tube
• Sort each to the assigned sphere
color by a zip code
Multiplexing III, more advanced
• oops, the scheme below does not work
because different fluors will label the same
ball…….
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