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

Authors

  • Anuj Kumar Tyagi Department of Microbiology, Govt. Medical College, Kannauj, Uttar Pradesh, India. Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland and CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
  • Mary Boudal Khoshbeen Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland and CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
  • Patricia Huezo-Diaz Curtis Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland and CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
  • Chakradhara Rao S. Uppugunduri Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland and CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.
  • Marc Ansari Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland and CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

DOI:

https://doi.org/10.14440/jbm.2018.224

Keywords:

allele-specific PCR, exon, MGMT, rs2308321, rs113813075, genotyping

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. Genomic DNA of lymphoblastoid cell line obtained from the Coriell repository with known genotypes were used to standardize the genotyping procedure. The PCR products were analyzed by 3% Agarose gel electrophoresis and by DNA Screen Tape assay with the Agilent 4200 TapeStation. The allele-specific PCR assay described here is a suitable strategy for efficient and reliable genotyping for difficult variants. This method offers cost-effective strategy for genotyping in clinical cohort studies provided positive controls established by Sanger sequencing are available for the variant. 

Author Biography

Anuj Kumar Tyagi, Department of Microbiology, Govt. Medical College, Kannauj, Uttar Pradesh, India. Onco-Hematology Unit, Department of Pediatrics, Geneva University Hospitals, University of Geneva, Geneva, Switzerland and CANSEARCH Research Laboratory, Department of Pediatrics, Faculty of Medicine, University of Geneva, Geneva, Switzerland.

Department of Microbiology, Government Medical College, Kannauj, Uttar Pradesh (U.P), India. 

Lecturer/Assistant Professor

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Published

2018-06-07

How to Cite

1.
Tyagi AK, Khoshbeen MB, Curtis PH-D, Uppugunduri CRS, Ansari M. Development and validation of an allele-specific PCR assay for genotyping a promoter and exonic single nucleotide polymorphisms of MGMT gene. J Biol Methods [Internet]. 2018Jun.7 [cited 2022Aug.11];5(2):e92. Available from: https://jbmethods.org/jbm/article/view/224

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