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Glucocorticoid receptor HRM kit for SNPs detection

C-C / G-G C-G. C-C A-G G-G. G-G G-A A-A. A-A A-G G-G.

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Glucocorticoid receptor HRM kit for SNPs detection

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  1. C-C / G-G C-G C-C A-G G-G G-G G-A A-A A-A A-G G-G Glucocorticoid receptor HRM kit for SNPs detection Glucocorticoid (GC) hormones play a key role in metabolic and immunological homeostasis. They regulate many physiological processes and, due to their immunosuppressive and antinflammatory actions, synthetic GCs are widely employed in the treatment of a variety of immune system diseases. Glucocorticoid receptor (GR) belongs to nuclear receptors superfamily which are present in cytoplasm and act as transcription factors to regulate gene expression. BclI, N363S and ER22/23EK polymorphisms have a putative effect on GC sensitivity on the basis of the previously described clinical relevance and frequencies in caucasian population [ E.F. van Rossum et al., 2002 ]. The ER22/23EK polymorphism consists of two linked single-nucleotide mutations in codons 22 and 23 (exon 2 of the GR gene). This genetic variant causes a relative decrease in GCs sensitivity. Single nucleotide polymorphism N363S, is a point mutation in exon 2 that causes an asparagine to serine amino acid substitution in codon 363. The 363S allele has been associated with increased sensitivity to GCs. As extensively reported by literature, the clinically most relevant polymorphism of the GR gene is the BclI polymorphism. This mutation was recently identified as a C/G nucleotide localized in intron 2 and it has been associated with increased sensitivity to GCs. The Glucocorticoid receptor HRM kit represents an alternative PCR-based approach for the simultaneous identification of BclI, N363S and ER22/23EK polymorphisms located within the GR gene by High Resolution Melt (HRM). PRODUCT DESCRIPTION –The Glucocorticoid receptor HRM Kit provide an easy to use master mixes and synthetics controls for simultaneous amplification and detection by HRM analysis of BclI, N363S, ER22/23EK polymorphisms. HRM analysis is performed after real-time PCR. Amplification and dissociation's plots are obtained by use of EVA GREEN intercalating dye. Up to 3 different SNPs can be simultaneously detected with the same run by their dissociation profile. This assay is also suitable for inexpensive high throughput screening studies being HRM technology the most cost-efficient method for SNPs detection today available.The kit has been validated with Rotor Gene 6000 Instrument (Corbett Research) and Rotor Gene Q (QIAGEN) UP TO 3 SNPs FOR GLUOCCORTICOID RESPONSE STUDY DATA ANALYSIS -Due to the comparison between controlsand unknown samples HRM profiles the instrument’s software performs an automatic genotyping showing a confidence level for each sample. The identity of each PCR product obtained from unknown samples will be automatically assigned comparing its melting temperature with the Tm of the positive controls. Due to the high similarity melting profile for C-C and G-G amplicons the BclI mutation can be discriminated only by a spiking experiment performing a contamination of an unknown sample with a known control. ER22/23EK Fig.1.B Fig.1.A • Fig.1 : Graphical visualizations for the identification of the ER22/23EK mutation. • Melting curves show heteroduplexes formation in the G-A population (in blue) and temperature shift between G-G and A-A genotypes (green and red curves, respectively). • B) Normalized melting curves of the three genotypes N363S Fig.2.B Fig.2.A • Fig. 2: Graphical visualizations for the identification of the N363S mutation. • Melting curves show heteroduplexes formation in the A-G population (in blue) and temperature shift between A-A and G-G genotypes (green and red curves, respectively). • B) Normalized melting curves of the three genotypes. Fig.3.A BclI - before spiking experiment Fig.3.B • Fig. 3: Graphical visualizations for the identification of the BclI mutation before spiking experiment • Melting curves show C-G genotype population (in blue) and the superimposed curves of C-C and G-G genotypes (green). Fig.1. BclI - after spiking experiment Fig.4.A Fig.4.A Fig.4.B Fig.4.B • Fig. 4: Graphical visualizations for the identification of the BclI mutation after spiking experiment • Melting curves show C-G genotype (in blue) and G-G genotype (in red) after spiking experiment. Green lines represent C-C genotype. All graphics have been developed by Rotor- Gene 6000 Series Software version 1.7 (Corbett Research)

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