For Research Use Only. Not for use in diagnostic procedures. Others LightMix Kit HFE H63D S65C C282Y RUO LightMix® Kit HFE H63D S65C C282Y 100 1377683945183 This product is not a Roche product, Roche Global Customer Support is not offering any Support. For any additional information and support please ONLY contact TIB MolBiol directly. Furthermore we can not guarantee that all available information for this product is up to date. PID00000651 Detection Kit for HFE gene DNA variations p.H63D, p.S65C and p.C282Y This kit detects and identifies the SNP variants p.H63D, p.S65C, and p.C282Y in the HFE gene from human genomic DNA extracts. A 284 bp long PCR fragment containing the c.845G>A (C282Y) polymorphism is analyzed using a LC640 oligomer (640 channel) matching the mutant 282Y allele. A 163 bp long PCR fragment containing the c.187C>G (63 H/D) and c.193A>T (65 S/C) polymorphisms is analyzed using a SimpleProbe® 519 oligomer (530 channel) which matches the mutant 63D allele. A melting curve analysis is used to determine the genotype. For use on Roche LightCycler® systems. en Using PCR methodology, two fragments of the HFE gene are amplified simultaneously with specific oligonucleotide primers. Fluorescent labeled probes are used to identify the PCR product and to determine the genotype by performing a melting curve analysis.The probe binds to a part of the amplified fragment spanning the mutation site. Any mismatch covered by the probe destabilizes the hybrid. During the melting curve analysis the temperature is slowly increased. The probe melts off at a specific melting temperatures causing the fluorescence to decrease.Exon 4: Variant p.C282YA 284 bp long PCR fragment containing the c.845G>A (C282Y) polymorphism is analysed using a LightCycler® Red 640 red oligomer matching the mutant 282Y allele. In the melting curve analysis the 282Y samples display a higher temperature than the wild type C282 allele.Other variants, e.g. 281M or 283P yield different melting temperatures.Exon 2: Variants p.H63D and p.S65CA 163 bp long PCR fragment containing the c.187C>G (63 H/D) and c.193A>T (65 S/C) polymorphisms is analyzed using a SimpleProbe® 519 oligomer which matches the mutant 63D allele.The probe binds to a part of the amplified fragment starting from amino acid 58 Phe and passing beyond the mutation sites.Figure 1: Partial gene sequence.In the melting curve analysis the 63D samples display a higher temperature than the wild type allele H63, while the 65C samples have the lowest melting temperature. Other variants yield different melting temperatures.Reading of the genotype results is based on the melting temperatures compared to the supplied standards. If permitted by the instrument software reading of the genotype results can be achieved by the automated genotyping (instrument- dependent: software module 'Melt Curve Genotyping').Automated genotyping results must be reviewed by eye for deviating curves and different melting point temperatures. In case that automated typing fails to report consistent genotype results, the genotype must be deducted from the melting temperatures following the criteria described in chapter 8.Automated genotyping might fail in case that the supplied standards generate much higher signals than average extracted human samples. In this case use known (pre- typed) extracted samples as references for teaching the genotypes. en Analytical SensitivityDetection of serial dilutions of several heterozygous human genomic DNA has revealed that the limit of detection of the present kit is 250 copies DNA (1.5 ng).Analytical SpecificityThe specificity to the target gene and the suitability of the PCR amplification employed in the present kit was demonstrated by comparison with the results obtained by direct sequencing of the amplicon for HFE H63D S65C and the amplicon for HFE C282Y. en

LightMix^® Kit HFE H63D S65C C282Y

Detection Kit for HFE gene DNA variations p.H63D, p.S65C and p.C282Y

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LightMix^® Kit HFE H63D S65C C282Y

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