PCR Conditions for HFE C282Y: Lack of Effect of 5569G/A Polymorphism with 55 °C Annealing
Open Access
- 1 February 2000
- journal article
- Published by Oxford University Press (OUP) in Clinical Chemistry
- Vol. 46 (2) , 303
- https://doi.org/10.1093/clinchem/46.2.303a
Abstract
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 site was designed (4).Keywords
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