High resolution melting (HRM) analysis for the detection of ER22/23EK, BclI, and N363S polymorphisms of the glucocorticoid receptor gene

Maltese, Paolo Enrico; Canestrari, Emanuele; Palma, Linda; Ruzzo, Annamaria; Corini, Francesco; Menotta, Michele; Andreoni, Francesca; Annese, Vito; Magnani, Mauro

doi:10.1016/j.jsbmb.2009.01.012

Cited by 22 publications

(19 citation statements)

References 35 publications

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“…HRM is usually applied to the analysis of single SNPs in much smaller fragments (13)(14)(15)29). The ability of HRM to correctly resolve the 47 flaA 620-bp fragment alleles included in this study was somewhat unexpected.…”

Section: Discussionmentioning

confidence: 90%

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Merchant-Patel

Blackall²,

Templeton³

et al. 2010

Appl Environ Microbiol

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The highly variable flagellin-encoding flaA gene has long been used for genotyping Campylobacter jejuni and Campylobacter coli. High-resolution melting (HRM) analysis is emerging as an efficient and robust method for discriminating DNA sequence variants. The objective of this study was to apply HRM analysis to flaA-based genotyping. The initial aim was to identify a suitable flaA fragment. It was found that the PCR primers commonly used to amplify the flaA short variable repeat (SVR) yielded a mixed PCR product unsuitable for HRM analysis. However, a PCR primer set composed of the upstream primer used to amplify the fragment used for flaA restriction fragment length polymorphism (RFLP) analysis and the downstream primer used for flaA SVR amplification generated a very pure PCR product, and this primer set was used for the remainder of the study. Eighty-seven C. jejuni and 15 C. coli isolates were analyzed by flaA HRM and also partial flaA sequencing. There were 47 flaA sequence variants, and all were resolved by HRM analysis. The isolates used had previously also been genotyped using single-nucleotide polymorphisms (SNPs), binary markers, CRISPR HRM, and flaA RFLP. flaA HRM analysis provided resolving power multiplicative to the SNPs, binary markers, and CRISPR HRM and largely concordant with the flaA RFLP. It was concluded that HRM analysis is a promising approach to genotyping based on highly variable genes.Campylobacter jejuni and Campylobacter coli are the most common causes of human bacterial gastroenteritis in industrialized countries (21). The flagellin-encoding genes flaA and flaB share 95% sequence homology and are arranged in tandem (9,20). While flaA gene expression appears critical for motility, colonization, and pathogenesis, this is not the case for flaB, which is thought to be a largely nonfunctional reservoir of genetic variation that can increase the diversity of flaA by recombination and so assist the cell in evading host immune responses (1,7,8,28).The flaA gene is commonly used for typing C. jejuni and C. coli. Two methods have gained wide acceptance: flaA restriction fragment length polymorphism (RFLP) (19) and flaA short variable region (SVR) sequencing (16). The flaA RFLP technique involves PCR amplification of the entire flaA gene followed by RFLP of the PCR product (19). Sequencing the SVR of the flaA gene was developed as a more streamlined and portable alternative to flaA RFLP protocols (16), and sequence variants are compiled at a central website (http://pubmlst.org/campylobacter /flaA/) (10). While both of these methods are very effective, they have several disadvantages: the RFLP approach is multistep, because the PCR product must be cleaved with a restriction enzyme, and the fragments must be subsequently resolved by electrophoresis (33). Also, there are many changes in the sequence that will not alter the sizes of the restriction fragments. SVR sequencing is also multistep; the targeted region is small, which limits resolving power; and DNA sequencing requires expensive equipment ...

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Section: Discussionmentioning

confidence: 90%

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Merchant-Patel

Blackall²,

Templeton³

et al. 2010

Appl Environ Microbiol

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“…Moreover, HRM is, as yet, affected by low resolution, limiting, in particular, discrimination of homozygous mutants from homozygous wild-type samples, as we have previously described. 25 Nonetheless, HRM analysis avoids the excessive sample handling, long times and high costs of other techniques. In addition, considering the bioinformatics component of this technique, it is likely that future implementation will make the HRM analysis an even more powerful method.…”

Section: Discussionmentioning

confidence: 99%

“…HRM analysis enables the distinction of homozygous wild-type, homozygous mutant and heterozygous mutant genotypes, as it is shown for the rs3800373, rs1360780 and rs4713916 mutations (See Supplementary Figures 1-3). This technique was used here to develop a method for the simultaneous genotyping of BclI, N363S, ER22/23EK polymorphisms located within the GR gene, 25 and of rs3800373, rs1360780 and rs4713916 polymorphisms located within FKBP5 gene using the same reaction mixture (data not shown). Thus, our system enables the simultaneous detection of six different polymorphisms related to the response to GCs.…”

Section: Validation Of Fkbp5 Snps Assaymentioning

confidence: 99%

“…26 PCR primers were designed by Diatheva (Diatheva S.r.l., Fano, Italy) according to the same parameters previously described for GR polymorphisms, 25 avoiding hairpins and primer-dimer formation and maintaining the amplicon length within the 80-150 bp range. Assembly of PCR reactions, cycling and HRM analyses, specifically designed for the allelic discrimination of all SNPs in a single run on the Rotor-Gene 6000TM were performed as described elsewhere.…”

Section: Validation Of Fkbp5 Snps Assaymentioning

confidence: 99%

“…Assembly of PCR reactions, cycling and HRM analyses, specifically designed for the allelic discrimination of all SNPs in a single run on the Rotor-Gene 6000TM were performed as described elsewhere. 25 Briefly, the reaction mixture contained 10 ng of genomic DNA,…”

Section: Validation Of Fkbp5 Snps Assaymentioning

confidence: 99%

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Glucocorticoid resistance in Crohn's disease and ulcerative colitis: an association study investigating GR and FKBP5 gene polymorphisms

et al. 2011

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The aim of this study is to investigate the role of single-nucleotide polymorphisms (SNPs) of the glucocorticoid receptor (GR) and of the related co-chaperone FKBP5 genes in the development of glucocorticoid (GC) resistance in Crohn's disease (CD) and ulcerative colitis (UC) patients. We have developed a high-resolution DNA melting method that allows simultaneous identification of GR (BclI, N363S and ER22/23EK) and FKBP5 (rs3800373, rs1360780 and rs4713916) polymorphisms. Genotype frequencies were determined in 100 consecutive CD and 100 UC patients under GCs therapy (50 responders and 50 resisters). The variation of FKBP5 polymorphism rs4713916 (G/A), in the putative promoter region of FKBP5, is significantly associated with resistance to GC treatment in CD (responder ¼ 17% versus resister ¼ 35%; P ¼ 0.0043). No significant differences were found in UC patients. If these preliminary findings will be confirmed, the combination of GR and FKBP5 mutational analyses could help to identify subgroups of CD patients with higher chances to benefit from GC treatment.

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Polymorphisms of the glucocorticoid receptor gene: impact on clinical outcome of multiple myeloma

et al. 2011

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High resolution melting (HRM) analysis for the detection of ER22/23EK, BclI, and N363S polymorphisms of the glucocorticoid receptor gene

Cited by 22 publications

References 35 publications

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Campylobacter jejuni and Campylobacter coli Genotyping by High-Resolution Melting Analysis of a flaA Fragment

Glucocorticoid resistance in Crohn's disease and ulcerative colitis: an association study investigating GR and FKBP5 gene polymorphisms

Polymorphisms of the glucocorticoid receptor gene: impact on clinical outcome of multiple myeloma

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