Clinical Chemistry
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Clinical Chemistry 43: 1336-1341, 1997;
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(Clinical Chemistry. 1997;43:1336-1341.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

A homogeneous method for genotyping with fluorescence polarization

Neil J. Gibson, Helen L. Gillard, David Whitcombe, Richard M. Ferrie, Clive R. Newton and Stephen Littlea

a Author for correspondence. Fax +44 1 606 49366.

We combined the amplification refractory mutation system (ARMSTM) and fluorescence polarization (FP) to give a homogeneous genomic DNA genotype analysis method. Oligonucleotide probes labeled with the fluorescein dyes fluorescein isothiocyanate and 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein and the rhodamine dye 6-carboxyrhodamine were included in amplification mixes and were annealed to PCR products after amplification. Hybridization was accompanied by an increase in the FP of the probe. We demonstrated homogeneous genotyping by analyzing human DNA samples for {Delta}F508 mutation status of the cystic fibrosis transmembrane conductance regulator gene. The genotypes determined with the method described herein were in full agreement with those obtained by the conventional application of ARMS. We also demonstrated the simultaneous detection of two PCR products in a single reaction. The assay method described is homogeneous and so obviates the necessity to open reaction vessels after amplification. This therefore eliminates PCR carryover contamination.


Key Words: indexing terms: DNA amplification • cystic fibrosis • genetic variants




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