Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of

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Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of

MGMT gene

TYAGI, Anuj Kumar, et al .

Abstract

DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) specifically remove the methyl/alkyl group from the O6-position of guanine and restore the guanine to its normal form without causing DNA strand breaks. Relationship between MGMT activity and resistance to alkylating therapeutic agents is well established. Non-availability of simple, cost-effective and efficient methods of genotyping may hinder investigations on genotype-phenotype associations. No simple genotyping procedures such as allele-discrimination Taqman Assays were available for two genetic variations in MGMT gene that had previously demonstrated to be affecting its function and expression. These two variants were included to genotype in a clinical study (Clinicaltrail.gov ID: NCT01257854). Hence, the present study is aimed at developing, validating a rapid and simple allele-specific PCR method that genotypes exonic variant rs2308321 (c.520A>G) and a promoter variant rs113813075 (c.-459C>A) with standard PCR instruments. Web-based allele-specific (AS) primer design application called web-based allele-specific primer was used to design primers. [...]

TYAGI, Anuj Kumar, et al . Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene. Journal of Biological Methods , 2018, vol. 5, no. 2, p. e92

PMID : 31453242

DOI : 10.14440/jbm.2018.224

Available at:

http://archive-ouverte.unige.ch/unige:128695

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Title: Development and validation of an Allele-Specific PCR Assay for Genotyping a Promoter and Exonic single nucleotide polymorphisms of O

6

-methylguanine-DNA methyltransferase (MGMT) gene.

Names of Authors: Anuj Kumar Tyagi*

1,2,3

, Mary Boudal Khoshbeen

2,3

, Patricia Huezo-Diaz Curtis

2,3

, Chakradhara Rao Satyanarayana Uppugunduri

2,3

& Marc Ansari

2,3

Authors’ Affiliations:

1

Current affliation: Department of Microbiology, Govt. Medical College, Kannauj, Uttar Pradesh, India.

2

CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

3

Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland.

*Corresponding Author: Dr. Anuj Kumar Tyagi, Ph.D; Department of Microbiology; Govt. Medical College, Kannauj, Uttar Pradesh; India ; E.mail: tyagi.aiims@gmail.com

Supplementary material

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rs2308321 (NA-12762; A/A) rs2308321 (NA-108959; A/A) rs2308321 (NA-07034; G/A)

rs113813075 (NA-10847; A/A) rs113813075 (NA-6985; C/C)

Supplementary Figure 1: electropherogram showing the confirmation of Exonic SNP (rs2308321)

and Promoter SNP (rs113813075) Genotypes by Sanger sequencing.

Figure

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