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