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Clinical Chemistry 46: 303, 2000;
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(Clinical Chemistry. 2000;46:303.)
© 2000 American Association for Clinical Chemistry, Inc.

Letters

PCR Conditions for HFE C282Y: Lack of Effect of 5569G/A Polymorphism with 55 °C Annealing

Christine Y.B. Chin1,a

a Author for correspondence. Fax 61-8-9346-3882; e-mail christine.chin@health.wa.gov.au.

John P. Beilby1
Enrico Rossi1
Gary P. Jeffrey2

1 Biochemistry Section, Pathcentre, QE II Medical Centre, Nedlands, Western Australia 6009

2 Department of Medicine, University of Western Australia, Nedlands, Western Australia 6009


To the Editor:

The HFE gene mutation designated C282Y (g.5474G->A; GenBank accession no. Z92910) is usually detected by the primers described by Feder et al. (1) in a PCR to amplify genomic DNA followed by restriction enzyme cleavage based on the production of either a SnaB1 (2) or a Rsa1 (3) cut site. A polymorphism (5569G/A) located five nucleotides within the 3' end of the binding site for the antisense primer has been reported to lead to false-positive results for C282Y homozygosity (4). The false-positive result occurs in analysis of a C282Y heterozygote compound with the 5569G/A polymorphism because of selective amplification of the C282Y-containing allele. A new antisense primer (5'-TACCTCCTCAGGCACTCCTC-3') that did not bind the 5569G/A . . . [Full Text of this Article]


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