Simultaneous genotyping of GSTT1 and GSTM1 null polymorphisms by melting curve analysis in presence of SYBR Green I.

2.50
Hdl Handle:
http://hdl.handle.net/10146/114895
Title:
Simultaneous genotyping of GSTT1 and GSTM1 null polymorphisms by melting curve analysis in presence of SYBR Green I.
Authors:
Marin, Fatima; Garcia, Nadia; Munoz, Xavier; Capella, Gabriel; Gonzalez, Carlos A.; Agudo, Antonio; Sala, Nuria
Abstract:
Due to their ability to metabolize xenobiotics, glutathione S-transferases (GSTs) play an important role in cellular protection. GST family members mu (GSTM1) and theta (GSTT1) exhibit a common polymorphism that results in the complete deletion of the gene (null allele). Homozygous deletions, which result in the absence of the enzyme, are considered a risk factor for several diseases, including cancer. We report a simple, low cost, and high throughput assay for the simultaneous analysis of the GSTM1 and GSTT1 null polymorphisms in a single step. The assay is based on multiplex real-time PCR in the presence of SYBR Green I and genotype discrimination by melting curve analysis in a LightCycler. We have genotyped 792 samples to compare this new approach with conventional PCR followed by gel electrophoresis. Comparison of the methods gave a good agreement, with kappa values of 0.88 for GSTM1 and 0.64 for GSTT1. Reanalysis of discrepant samples indicated that absence of amplification of the larger GSTT1 fragment by conventional PCR accounted for most of the discrepancies. Moreover, the improved amplification efficiency of the real-time PCR results in a significant reduction of missing values. Due to its simplicity and low cost, this assay is well suited for the rapid analysis of GST-null genotypes in studies that involve large number of samples.
Citation:
J. Mol. Diagn. 2010, 12 (3):300-304
Journal:
The Journal of Molecular Diagnostics : JMD
Issue Date:
May-2010
URI:
http://hdl.handle.net/10146/114895
DOI:
10.2353/jmoldx.2010.090076
PubMed ID:
20203006
Additional Links:
http://jmd.amjpathol.org/cgi/content/full/12/3/300
Type:
Article
Language:
en
ISSN:
1943-7811
Sponsors:
The authors are members of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a Network of Excellence of the 6th EU Framework Programme (FP6, FOODCT- 2005-513 943), and of the ISCIII Spanish Ministry of Health network RTICCC (ISCIII DR06/0020).
Appears in Collections:
Articles

Full metadata record

DC FieldValue Language
dc.contributor.authorMarin, Fatimaen
dc.contributor.authorGarcia, Nadiaen
dc.contributor.authorMunoz, Xavieren
dc.contributor.authorCapella, Gabrielen
dc.contributor.authorGonzalez, Carlos A.en
dc.contributor.authorAgudo, Antonioen
dc.contributor.authorSala, Nuriaen
dc.date.accessioned2010-11-08T10:14:51Z-
dc.date.available2010-11-08T10:14:51Z-
dc.date.issued2010-05-
dc.identifier.citationJ. Mol. Diagn. 2010, 12 (3):300-304en
dc.identifier.issn1943-7811-
dc.identifier.pmid20203006-
dc.identifier.doi10.2353/jmoldx.2010.090076-
dc.identifier.urihttp://hdl.handle.net/10146/114895-
dc.description.abstractDue to their ability to metabolize xenobiotics, glutathione S-transferases (GSTs) play an important role in cellular protection. GST family members mu (GSTM1) and theta (GSTT1) exhibit a common polymorphism that results in the complete deletion of the gene (null allele). Homozygous deletions, which result in the absence of the enzyme, are considered a risk factor for several diseases, including cancer. We report a simple, low cost, and high throughput assay for the simultaneous analysis of the GSTM1 and GSTT1 null polymorphisms in a single step. The assay is based on multiplex real-time PCR in the presence of SYBR Green I and genotype discrimination by melting curve analysis in a LightCycler. We have genotyped 792 samples to compare this new approach with conventional PCR followed by gel electrophoresis. Comparison of the methods gave a good agreement, with kappa values of 0.88 for GSTM1 and 0.64 for GSTT1. Reanalysis of discrepant samples indicated that absence of amplification of the larger GSTT1 fragment by conventional PCR accounted for most of the discrepancies. Moreover, the improved amplification efficiency of the real-time PCR results in a significant reduction of missing values. Due to its simplicity and low cost, this assay is well suited for the rapid analysis of GST-null genotypes in studies that involve large number of samples.en
dc.description.sponsorshipThe authors are members of ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a Network of Excellence of the 6th EU Framework Programme (FP6, FOODCT- 2005-513 943), and of the ISCIII Spanish Ministry of Health network RTICCC (ISCIII DR06/0020).en
dc.language.isoenen
dc.relation.urlhttp://jmd.amjpathol.org/cgi/content/full/12/3/300en
dc.subjectOrganic Chemicalsen
dc.subjectGenotypeen
dc.subjectGlutathione Transferaseen
dc.subjectPolymorphism, Geneticen
dc.subjectHumansen
dc.subjectPolymerase Chain Reactionen
dc.subjectElectrophoresisen
dc.subject.meshElectrophoresis-
dc.subject.meshGenotype-
dc.subject.meshGlutathione Transferase-
dc.subject.meshHumans-
dc.subject.meshNucleic Acid Denaturation-
dc.subject.meshOrganic Chemicals-
dc.subject.meshPolymerase Chain Reaction-
dc.subject.meshPolymorphism, Genetic-
dc.titleSimultaneous genotyping of GSTT1 and GSTM1 null polymorphisms by melting curve analysis in presence of SYBR Green I.en
dc.typeArticleen
dc.identifier.journalThe Journal of Molecular Diagnostics : JMDen

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