Logo Medical Science Monitor

Call: +1.631.470.9640
Mon - Fri 10:00 am - 02:00 pm EST

Contact Us

Logo Medical Science Monitor Logo Medical Science Monitor Logo Medical Science Monitor

09 January 2002

Detection of gsp somatic mutation through direct sequencing of heteroduplex alleles disclosed by denaturing gradient gel electrophoresis.

Maria C Barisson Villares Fragoso, Valeria S Lando, Ana C Latronico, Eliana T Salgado Frazzatto, Alan J Russell, Berenice B Mendonca

Med Sci Monit 2002; 8(1): BR15-18 :: ID: 420977

Abstract

BACKGROUND: The identification of somatic mutations in tissues is oftendifficult when the number of normal alleles in the tissue far exceeds the number of mutant ones. We foundthat the identification of gsp mutation was not possible by direct sequencing and present a new approachthat improves the identification of gsp somatic mutations. MATERIAL/METHODS: Genomic DNA was extractedfrom frozen tissue of a human ovarian stromal Leydig cell tumor. Exons 8 and 9 of the Gsa gene were amplifiedby PCR and despite the abnormal migration pattern at this first DGGE, direct sequencing of the PCR productdid not reveal mutations, probably due to the small amount of mutant alleles. To improve this amount,the PCR products were re-amplified using as template the excised products of the mutant homoduplex andheteroduplex bands obtained at the first DGGE. RESULTS: This approach resulted in the enhancing of themutant homoduplex bands whereas the heteroduplex bands remained unchanged at the second DGGE. Directsequencing of the second round PCR clearly identified the mutation R201C in the ovarian Leydig cell tumor.CONCLUSIONS: We have demonstrated a relatively rapid, convenient and reliable method to improve gsp somaticmutation detection combining a second DGGE of the PCR products obtained from the heteroduplexes and mutanthomoduplex bands disclosed in a first DGGE followed by direct sequencing.

Keywords: Alleles, Electrophoresis, Polyacrylamide Gel, GTP-Binding Protein alpha Subunits, Gs, Heterozygote, Mutation, Nucleic Acid Heteroduplexes, Polymerase Chain Reaction, Research Support, Non-U.S. Gov, Sequence Analysis, DNA

Add Comment 0 Comments

Editorial

01 May 2024 : Editorial  

Editorial: First Regulatory Approval for Adoptive Cell Therapy with Autologous Tumor-Infiltrating Lymphocytes (TILs) – Lifileucel (Amtagvi)

Dinah V. Parums

DOI: 10.12659/MSM.944927

Med Sci Monit 2024; 30:e944927

In Press

07 May 2024 : Clinical Research  

Treatment of AVN-Induced Proximal Pole Scaphoid Nonunion Using a Fifth and Fourth Extensor Compartmental Ar...

Med Sci Monit In Press; DOI: 10.12659/MSM.944553  

0:00

16 Mar 2024 : Clinical Research  

Diagnostic Efficiency of ACR-TIRADS Score for Differentiating Benign and Malignant Thyroid Nodules of Vario...

Med Sci Monit In Press; DOI: 10.12659/MSM.943228  

0:00

08 May 2024 : Clinical Research  

Effect of Individualized PEEP Guided by Driving Pressure on Diaphragm Function in Patients Undergoing Lapar...

Med Sci Monit In Press; DOI: 10.12659/MSM.944022  

0:00

21 Mar 2024 : Clinical Research  

Impact of Serum Vitamin D, B6, and B12 and Cognitive Functions on Quality of Life in Peri- and Postmenopaus...

Med Sci Monit In Press; DOI: 10.12659/MSM.943249  

Most Viewed Current Articles

17 Jan 2024 : Review article  

Vaccination Guidelines for Pregnant Women: Addressing COVID-19 and the Omicron Variant

DOI :10.12659/MSM.942799

Med Sci Monit 2024; 30:e942799

0:00

14 Dec 2022 : Clinical Research  

Prevalence and Variability of Allergen-Specific Immunoglobulin E in Patients with Elevated Tryptase Levels

DOI :10.12659/MSM.937990

Med Sci Monit 2022; 28:e937990

0:00

16 May 2023 : Clinical Research  

Electrophysiological Testing for an Auditory Processing Disorder and Reading Performance in 54 School Stude...

DOI :10.12659/MSM.940387

Med Sci Monit 2023; 29:e940387

0:00

01 Jan 2022 : Editorial  

Editorial: Current Status of Oral Antiviral Drug Treatments for SARS-CoV-2 Infection in Non-Hospitalized Pa...

DOI :10.12659/MSM.935952

Med Sci Monit 2022; 28:e935952

0:00

Your Privacy

We use cookies to ensure the functionality of our website, to personalize content and advertising, to provide social media features, and to analyze our traffic. If you allow us to do so, we also inform our social media, advertising and analysis partners about your use of our website, You can decise for yourself which categories you you want to deny or allow. Please note that based on your settings not all functionalities of the site are available. View our privacy policy.

Medical Science Monitor eISSN: 1643-3750
Medical Science Monitor eISSN: 1643-3750