Clinical Chemistry
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Clinical Chemistry 47: 1373-1377, 2001;
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(Clinical Chemistry. 2001;47:1373-1377.)
© 2001 American Association for Clinical Chemistry, Inc.

Articles

Genotyping Single-Nucleotide Polymorphisms by the Invader Assay with Dual-Color Fluorescence Polarization Detection

Tony M. Hsu1, Scott M. Law2, Shenghui Duan1, Bruce P. Neri2 and Pui-Yan Kwok1a

1 Division of Dermatology, Washington University School of Medicine, St. Louis, MO 63110.

2 Third Wave Technologies, Inc., 502 S. Rosa Rd., Madison, WI 53719.

aAddress correspondence to this author at: Division of Dermatology, Washington University, 660 S. Euclid Ave., Box 8123, St. Louis, MO 63110. E-mail kwok{at}genetics.wustl.edu.

Background: The PCR-Invader® assay is a robust, homogeneous assay that has been shown to be highly sensitive and specific in genotyping single-nucleotide polymorphism (SNP) markers. In this study, we introduce two changes to improve the assay: (a) we streamline the PCR-Invader method by assaying both alleles for each SNP in one reaction; and (b) we reduce the cost of the method by adopting fluorescence polarization (FP) as the detection method.

Methods: PCR product was incubated with Invader oligonucleotide and two primary probes at 93 °C for 5 min. Signal probes corresponding to the cleaved flaps of the primary probes [labeled with fluorescein and 6-carboxytetramethylrhodamine (TAMRA) dye] and Cleavase® VIII enzyme (a flap endonuclease) were then added to the mixture. This reaction mixture was incubated at 63 °C for 5 min. FP measurements were made with a fluorescence plate reader.

Results: Eighty-eight individuals were genotyped across a panel of 10 SNPs, using PCR product as template, for a total of 880 genotypes. An average "no call" rate of 3.2% was observed after first round of experiments. PCR products were remade in those samples that failed to produce any genotype in the first round, and all gave clear-cut genotypes. When the genotypes determined by the PCR-Invader assay and template-directed dye-terminator incorporation assay with FP were compared, they were in 100% concordance for all SNP markers and experiments.

Conclusions: The improvements introduced in this study make PCR-Invader assay simpler and more cost-effective, and therefore more suitable for high-throughput genotyping.




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