John M. S. Bartlett.pdf - Bio-Nica.info

124 Iannone et al.
Fig. 1. Diagram of microsphere-based SNP genotyping by OLA or SBCE. Allele discrimination
by OLA or SBCE uses PCR-amplified genomic DNA that encompasses the SNP (target
DNA), synthetic capture oligonucleotide probe (capture probes extend up to the polymorphic
base for OLA and end prior to the polymorphic base for SBCE), and a fluorescent reporter.
Each capture probe contains both a sequence that is complementary to the target sequence and a
unique 25-base ZipCode sequence that will link the genotyping result to a specific microsphere
population. For OLA, the reporter is a short target-complementary oligonucleotide sequence
that ends with a fluorescein molecule. If there is base pairing between the reporter and capture
probe, DNA ligase will covalently couple the fluorescent reporter to the capture molecule. For
the SBCE example shown above, the reporter is a fluorochrome or biotin-coupled ddTTP (or
ddUTP) or ddCTP (each labeled ddNTP is used in a different tube). The DNA polymerase
extends the capture probe by one base. In each case, the probes are thermally cycled to amplify
the signal on the capture probes. A suspension of cZipCode-coupled microsphere populations
is added and the capture probes are hybridized to the microspheres through their ZipCode tails.
After washing, the fluorescent profiles of the microspheres are analyzed by flow cytometry. The
OLA example shows a multiplexed reaction of a single SNP using two microsphere populations,
one for each allele. More extensive multiplexing of SNPs can be conducted by combining more
probes with complimentary microsphere populations. The SBCE reaction shows two uniplexed
reactions (one for each allele). The SBCE reaction may be multiplexed by combining probes
for different SNPs and assaying all A or G alleles in a single tube.
The three fluorescent colors associated with each microsphere (two to identify the
microsphere population and one for the genotyping result) are determined by flow
cytometry. We describe two different flow cytometric systems for SNP genotyping.
The first uses a standard bench-top cytometer (FACSCalibur, BD Biosciences, San
Jose, CA) with a 488-nm laser excitation source. Sixty-four individual popula-