Format

Send to

Choose Destination
Cytometry. 2000 Feb 1;39(2):131-40.

Multiplexed single nucleotide polymorphism genotyping by oligonucleotide ligation and flow cytometry.

Author information

1
Department of Molecular Sciences, Glaxo Wellcome Research Laboratories, Research Triangle Park, NC 27709-3398, USA. mai49583@glaxowellcome.com

Abstract

BACKGROUND:

We have developed a rapid, high throughput method for single nucleotide polymorphism (SNP) genotyping that employs an oligonucleotide ligation assay (OLA) and flow cytometric analysis of fluorescent microspheres.

METHODS:

A fluoresceinated oligonucleotide reporter sequence is added to a "capture" probe by OLA. Capture probes are designed to hybridize both to genomic "targets" amplified by polymerase chain reaction and to a separate complementary DNA sequence that has been coupled to a microsphere. These sequences on the capture probes are called "ZipCodes". The OLA-modified capture probes are hybridized to ZipCode complement-coupled microspheres. The use of microspheres with different ratios of red and orange fluorescence makes a multiplexed format possible where many SNPs may be analyzed in a single tube. Flow cytometric analysis of the microspheres simultaneously identifies both the microsphere type and the fluorescent green signal associated with the SNP genotype.

RESULTS:

Application of this methodology is demonstrated by the multiplexed genotyping of seven CEPH DNA samples for nine SNP markers located near the ApoE locus on chromosome 19. The microsphere-based SNP analysis agreed with genotyping by sequencing in all cases.

CONCLUSIONS:

Multiplexed SNP genotyping by OLA with flow cytometric analysis of fluorescent microspheres is an accurate and rapid method for the analysis of SNPs.

PMID:
10679731
[Indexed for MEDLINE]

Supplemental Content

Loading ...
Support Center