Regular Article
Screening for hereditary fructose intolerance mutations by reverse dot-blot

https://doi.org/10.1006/mcpr.1998.0208Get rights and content
Under a Creative Commons license
open archive

Abstract

An assay is described which is useful for genetic screening of the two most prevalent mutations that cause hereditary fructose intolerance (HFI). Both mutations lie within exon 5 of the aldolase B gene. Amplification of exon 5 from genomic DNA isolated from peripheral lymphocytes using biotinylated aldolase B-specific primers yields a biotin-tagged probe. This probe is hybridized to complementary poly(dT)-tailed allele specific oligonucleotides (ASOs) that are bound to a nylon membrane. The length of the ASOs, the amount bound to the membrane and the time of hybridization are optimized for discrimination of all four alleles under the same hybridization conditions. Detection of biotinylated amplified DNA is performed by creating an avidin-alkaline phosphatase complex and visualization by chemiluminescence. This assay can rapidly detect the two mutations, A149P and A174D, which cause >70% of HFI worldwide, and offers a rapid and sensitive assay that is much less invasive for the diagnosis of this often difficult to diagnose disorder.

Keywords

reverse dot-blot, polymerase chain reaction, allele specific oligonucleotide, non-radioactive, chemiluminescence, fructose intolerance, aldolase B.

Cited by (0)

f1

Author to whom all correspondence should be addressed at: Biology Department, Boston University, 5 Cummington StBoston, MA 02215, USA. Tel: +1 617 353 5310, Fax: +1 617 353 6340, e-mail: [email protected]