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Microsphere-Based SNP Genotyping 123
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Microsphere-Based Single Nucleotide
Polymorphism Genotyping
Marie A. Iannone, J. David Taylor, Jingwen Chen, May-Sung Li,
Fei Ye, and Michael P. Weiner
1. Introduction
Single nucleotide polymorphisms (SNPs) are single base differences in genomic
DNA (1). These single-base mutations, estimated to occur every 1000 bases, are
thought to represent the most common form of genetic variation in the human genome
(2). Several million SNPs have been identified (3). High-throughput analysis of these
variations will be required to understand their contribution to disease. We outline a
method that uses solution-based oligonucleotide ligation assay (OLA) (4) or singlebase
chain extension (SBCE) (5,6) for allele discrimination followed by hybridization
to fluorescently encoded microspheres. Flow cytometric analysis of the microspheres’
fluorescent profile yields rapid and accurate SNP genotyping (7,8).
Allele discrimination by OLA or SBCE uses (1) polymerase chain reaction (PCR)-
amplified genomic DNA that encompasses the SNP to be queried (‘target’ DNA); (2)
a synthetic “capture” oligonucleotide probe; and (3) a fluorescent “reporter” (Fig. 1).
In the allele discrimination reaction, an enzyme is used to covalently couple a reporter
molecule to the capture probe in a target-dependent fashion. Each capture probe contains
a sequence that is complementary to the target sequence and a ZipCode sequence that
will associate the genotype result with a specific microsphere population. In OLA, a
DNA ligase covalently couples the fluorescent reporter (a short oligonucleotide) to the
capture probe if the capture and reporter probes correctly match the target DNA. In
SBCE, a DNA polymerase adds a labeled dideoxynucleotide to the capture probe.
After thermal cycling to amplify the signal on the capture probes, the enzymatically
reacted capture probes are incubated with a suspension of up to 100 populations of
fluorescently encoded microspheres where each population is uniquely identified by
its fluorescent profile (9–11). Each microsphere population is covalently coupled to a
different complementary ZipCode (cZipCode) oligonucleotide sequence that associates
it with a capture probe and specific SNP allele. Flow cytometric analysis of the
microspheres simultaneously identifies both the microsphere type and the fluorescent
signal associated with the SNP genotype.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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