Gen Mutasyonunun Belirlenmesinde Rekombinaz Polimeraz Çoğaltım Tekniği Optimizasyonu Çalışmaları ve Sonuçları

Yıl 2023, Cilt: 23 Sayı: 6, 1363 - 1372, 28.12.2023

Beste Çağdaş , Sebastian Kerstıng

https://doi.org/10.35414/akufemubid.1322267

Öz

İnsan genlerindeki tek nükleotid polimorfizmleri (SNP'ler) çok önemli genetik değişikliklerdir ve PCR (polimeraz zincir reaksiyonu) veya NGS (yeni nesil dizileme), SNP analizinde yaygın olarak kullanılır. Yeni teknolojilerin ilerlemesi üzerine yapılan çalışmalar sayesinde izotermal nükleik asit amplifikasyon yaklaşımına ilgi artmıştır. Bu yöntemlerden biri olarak, rekombinaz polimeraz amplifikasyonu (RPA), hasta başı nükleik asit ölçümünde çekici bir alternatif oluşturmaktadır. Çalışma kapsamında seçilen hedef SNP'ler, PIK3CA gen bölgesinde (E542K, E545K) tanımlanan mutasyonlardır ve PIK3CA mutasyonlarının değerlendirildiği DNA örnekleri, MCF7, BT474 ve ayrıca SKBr3 kanser hücre hatlarından izole edilmiştir. RPA reaksiyon koşulları için optimizasyon çalışmalarında analiz süresi, sıcaklık, primer ve ayrıca magnezyum asetat konsantrasyonu parametreleri değerlendirilmiştir. RPA ürünlerinin en verimli şekilde elde edilebildiği reaksiyon optimizasyon çalışmaları sonuçlarına göre reaksiyon süresi 20 dk, sıcaklık 40°C, primer konsantrasyonu 10 µM ve MgOAc konsantrasyonu ise 140 mM olarak değerlendirilmiştir.

Anahtar Kelimeler

Tek Nükleotid Polimorfizmi, İzotermal Çoğaltım, Polimeraz Zincir Reaksiyonu, Rekombinaz Polimeraz Çoğaltım, Gen Mutasyonu, PIK3CA

Proje Numarası

1059B192001062

Optimization Studies and Results of Recombinase Polymerase Amplification Technique for Gene Mutation Detection

Öz

Single nucleotide polymorphisms (SNPs) in human genes are very significant genetic changes and PCR (polymerase chain reaction) or NGS (next-generation sequencing) are extensively employed in SNP analysis. Thanks to the studies on the progress of new technologies, interest in the isothermal nucleic acid amplification approach has increased. As one of these methods, recombinase polymerase amplification (RPA) represents an attractive option for point-of-care nucleic acid quantification. The target SNPs selected within the scope of the study are mutations identified in the PIK3CA gene region (E542K, E545K), and DNA samples which were evaluated about PIK3CA mutations were isolated from the cancer cells MCF7, BT474, and also SKBr3. The optimization studies for the RPA reaction conditions were carried out for parameters such as assay time, temperature, primer, and also magnesium acetate concentration. According to the results of the reaction optimization studies, in which the RPA products can be obtained in the most efficient way, the assay time was determined as 20 min; the temperature as 40°C; the primer concentration as 10 µM and the MgOAc concentration as 140 mM.

Anahtar Kelimeler

Single Nucleotide Polymorphism, Isothermal Amplification, Polymerase Chain Reaction, Recombinase Polymerase Amplification, Gene Mutation, PIK3CA

Destekleyen Kurum

TÜRKİYE BİLİMSEL VE TEKNOLOJİK ARAŞTIRMA KURUMU

Proje Numarası

1059B192001062

Teşekkür

We acknowledge the Scientific and Technological Research Council of Turkey (Tubitak 2219 Award # 1059B192001062) for Dr. Beste ÇAĞDAŞ. We are also grateful to the Fraunhofer Institute For Cell Therapy and Immunology for supporting this study at Dr. Markus von Nickisch-Rosenegk’s laboratory.

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