M Phillips1, C A Meadows, M Y Huang, A Millson, E Lyon. 1. Associated and Regional University Pathologist Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108, USA.
Abstract
BACKGROUND: Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm). METHODS AND RESULTS: The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine. CONCLUSIONS: Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.
BACKGROUND:Hemochromatosis is a common genetic disease, affecting one in every 200 individuals in the United States. A PCR assay was designed using fluorescent melting curve analysis to simultaneously detect the G845-->A (C282Y) and C187-->G (H63D) mutations. The G845-->A and C187-->G loci are distinguished by color, and mutant alleles are distinguished from wild type by probe melting temperature (Tm). METHODS AND RESULTS: The probe sets used two fluorophore pairs, fluorescein with LCRed 640 for G845-->A and fluorescein with LCRed 705 for C187-->G. The probes, complementary to the mutant allele, dissociate from the product at specific Tms. Wild-type alleles form mismatches with the probes, reducing the Tms by 6 degrees C (G845-->A) and 10 degrees C (C187-->G). One of 133 samples had a Tm shift 4 degrees C less than the wild-type Tm for the G845-->A locus. Sequencing confirmed the sample to be homozygous for G845-->A and heterozygous for a C-->A substitution at position 842 (C842-->A), substituting lysine for threonine. CONCLUSIONS: Multiplexing by color and Tm allows for simultaneous genotyping of each mutation. A novel base-pair alteration was detected in cis with a G845-->A mutation.
Authors: J N Feder; A Gnirke; W Thomas; Z Tsuchihashi; D A Ruddy; A Basava; F Dormishian; R Domingo; M C Ellis; A Fullan; L M Hinton; N L Jones; B E Kimmel; G S Kronmal; P Lauer; V K Lee; D B Loeb; F A Mapa; E McClelland; N C Meyer; G A Mintier; N Moeller; T Moore; E Morikang; C E Prass; L Quintana; S M Starnes; R C Schatzman; K J Brunke; D T Drayna; N J Risch; B R Bacon; R K Wolff Journal: Nat Genet Date: 1996-08 Impact factor: 38.330
Authors: E C Jazwinska; L M Cullen; F Busfield; W R Pyper; S I Webb; L W Powell; C P Morris; T P Walsh Journal: Nat Genet Date: 1996-11 Impact factor: 38.330
Authors: C E McLaren; V R Gordeuk; A C Looker; V Hasselblad; C Q Edwards; L M Griffen; J P Kushner; G M Brittenham Journal: Blood Date: 1995-09-01 Impact factor: 22.113
Authors: E Beutler; T Gelbart; C West; P Lee; M Adams; R Blackstone; P Pockros; M Kosty; C P Venditti; P D Phatak; N K Seese; K A Chorney; A E Ten Elshof; G S Gerhard; M Chorney Journal: Blood Cells Mol Dis Date: 1996 Impact factor: 3.039
Authors: C Bartolo; P E McAndrew; R C Sosolik; K A Cawley; S P Balcerzak; J T Brandt; T W Prior Journal: Arch Pathol Lab Med Date: 1998-07 Impact factor: 5.534
Authors: Holger Kirsten; Daniel Teupser; Jana Weissfuss; Grit Wolfram; Frank Emmrich; Peter Ahnert Journal: J Mol Med (Berl) Date: 2006-12-08 Impact factor: 4.599