Beads are good for you and for genotyping Fan et al. 2005. Biotechniques 39, 583 (Illumina) Chen et...
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Transcript of Beads are good for you and for genotyping Fan et al. 2005. Biotechniques 39, 583 (Illumina) Chen et...
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