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Beads are good for you
and for genotypingFan 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
…ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA…
TCGAAACTCATATCT CAGCGGGAGTTAGAC
DNA Ligase
3’ 5’
…ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA…TCGAAACTCATATC CAGCGGGAGTTAGAC
T
DNA Ligase
Oligonucleotide Ligation Assay (OLA)
amplify target by PCR
ligate oligos
detect ligation product
Primer extension
TCGAAACTCATATCT C
DNA Pol
3’
…ACGAGCTTTGAGTATAGTGTCGCCCTCAATCTGCCAA…TCGAAACTCATATC
…ACGAGCTTTGAGTATAGAGTCGCCCTCAATCTGCCAA…
C
C
One tube = one reaction = one assay?
C
C
C
C
SNP1
SNP2
SNP3
SNP4
Bead types
rosary beads
=
“test tube” bead
5 uM1 cM
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
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 fiber > 1 bead
1 bundle of 50k fibers -> 50k beads
96-well plate -> 0.5M beads
Illumina genotyping: Golden
gate
Illumina genotypes
Reviewing the product-bead relationship
AGTT
CAGG
home
TCAA
GTCC
address
TCAA
GTCC
snp1
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