Optimiztion of PCR reaction conditions for detection M235Tpolymorphism of Angiotensinogen gene in hypertensive patients

Authors

  • mais yousif Tishreen University
  • Faisal Radwan Tishreen University
  • Haitham yazagi Tishreen University
  • Eyas Alkhayer Tishreen University

Abstract

Background: Polymerase chain reaction (PCR) is a sensitive and expensive procedure, and its success requires many attempts. This study aims to develop optimization strategies to amplify a specific sequence in the AGT gene, to detect the M235T polymorphism associated hypertension.

Materials and methods: Genomic DNA was extracted by column method, and the PCR reaction was carried out using special primers, applying different conditions, and PCR products were detected using agarose gel electrophoresis.

Results: The optimized thermal cycler conditions for PCR reaction were: initial denaturation (temperature 94 °C, for 2 minutes), denaturation (94 °C, 30 seconds, 30 cycles), annealing (60 °C, 30 seconds, 30 cycles), elongation (72°C, 30 seconds, 30 cycles), final elongation (72°C, 5 minutes). The optimized concentrations and volumes of  reagents were as follows: master mix (10 μl), forward and reverse primer (3.5 μl of each at a concentration of  21 pmol), DNA template (2 μl at a concentration of 30-50 ng ⁄ μl).

Conclusion: Adjusting the annealing temperature, primer concentration, and thermal cycler conditions had the greatest role in adjusting the PCR conditions, and there was no need to make adjustments to the concentrations of other reagents, nor to use additional amounts of magnesium chloride or other additives to enhance the reaction.

Downloads

Published

2024-08-22

How to Cite

1.
يوسف م, فيصل رضوان, هيثم يازجي, إياس الخير. Optimiztion of PCR reaction conditions for detection M235Tpolymorphism of Angiotensinogen gene in hypertensive patients. Tuj-hlth [Internet]. 2024Aug.22 [cited 2024Nov.24];46(3):315-27. Available from: https://journal.tishreen.edu.sy/index.php/hlthscnc/article/view/17325

Issue

Vol. 46 No. 3 (2024): Tishreen University Journal for Research and Scientific Studies - Health Sciences Series

Section

Articles

License

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.

The authors retain the copyright and grant the right to publish in the magazine for the first time with the transfer of the commercial right to Tishreen University Journal for Research and Scientific Studies - Health Sciences Series

 Under a CC BY- NC-SA 04 license that allows others to share the work with of the work's authorship and initial publication in this journal. Authors can use a copy of their articles in their scientific activity, and on their scientific websites, provided that the place of publication is indicted in  Tishreen University Journal for Research and Scientific Studies - Health Sciences Series   .  The Readers have the right to send, print and subscribe to the initial version of the article, and the title of  Tishreen University Journal for Research and Scientific Studies - Health Sciences Series  Publisher

journal uses a CC BY-NC-SA license which mean

You are free to:

  • Adapt — remix, transform, and build upon the material
  •  
  • The licensor cannot revoke these freedoms as long as you follow the license terms.

 

  • Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use.
  • NonCommercial — You may not use the material for commercial purposes.
  • ShareAlike — If you remix, transform, or build upon the material, you must distribute your contributions under the same license as the original.
  • No additional restrictions — You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits.

Most read articles by the same author(s)

1 2 > >>