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Clinical Chemistry 43: 1151-1158, 1997;
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(Clinical Chemistry. 1997;43:1151-1158.)
© 1997 American Association for Clinical Chemistry, Inc.

Articles

Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry

Andreas Braun1, Daniel P. Little1 and Hubert Köster2,a

1 Sequenom Instruments, Mendelssohnstr. 15D, 22761 Hamburg, Germany.

2 Department of Biochemistry and Molecular Biology, University of Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
a Author for correspondence. Fax 49-40-89967610; e-mail abraun01{at}aol.com

A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which requires a single detection primer complementary to a region downstream of a target strand's variable site. On addition of a polymerase, three dNTPs, and the fourth nucleotide in dideoxy form, the primer is extended through the mutation region until the first ddNTP is incorporated; the mass of the extension products determines the composition of the variable site. Tests for five cystic fibrosis mutations, including two exon 11 sites measured in a biplex reaction, and for differentiating between three common alleles of the poly(T) tract at the intron 8 splice acceptor site of the CFTR gene are presented. All experimental steps required for PROBE are amenable to the high degree of automation desirable for a high-throughput diagnostic setting. Furthermore, it requires no fluorescent, chemiluminescent, or radioactive labeling; the mass signals measured offer a far more analytically definitive signal, leading in all cases to high-quality unambiguous and easily interpreted results.


Key Words: indexing terms: cystic fibrosis • PROBE • dideoxynucleotides




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Copyright © 1997 by the American Association for Clinical Chemistry.