Moleküler Tanıda Yüksek Çözünürlüklü Erime Yöntemi ve Klinik Önemi

Her geçen gün moleküler biyoloji alanında teknolojinin gelişmesiyle paralel, birçok yeni moleküler analiz yöntemleri ortaya çıkmaktadır. Biz de bu derlemede, moleküler tanıda önemli olduğu gösterilmiş olan Yüksek Çözünürlüklü Erime (High Resolution Melting) (HRM) analizini, uygulama alanlarını ve klinikte yapılmış çalışmalarla önemini anlatmayı amaçladık. HRM kapalı tüp sisteminde, Polimeraz Zincir Reaksiyonu (PZR: PCR) sonrasına dayalı genetik varyasyonları belirlemek için kullanılan yeni bir yöntemdir. HRM’nin çalışma prensibi nükleik asit örneklerinin erime davranışına dayanmaktadır. Çift zincirli DNA’nın denatürasyonu erime sıcaklığının artmasıyla oluşan floresan değişikliklerinin saptanmasıyla belirlenir. Wild type (yaban tip) ve heterozigot örneklerinin farklılıkları erime grafiklerinde kolaylıkla saptanabilmektedir. Bu yöntemde erime eğrisi analiziyle daha fazla bilgi ve detay elde edilebilmektedir. HRM analizi, örneklerin sekans, uzunluk, guanin sitozin (GC) içeriğine göre ayrımını yapabilmektedir. Popülasyonda yaygın görülen tek nükleotit değişikliklerinin (SNP) tespiti, hastalıklarla ilişkili gen mutasyon taramaları ve DNA metilasyon analizleri HRM yöntemi ile hızlı ve güvenilir bir şekilde tanımlanabilmektedir. PZR ürünlerindeki nükleotit dizi değişimleri ve çeşitli varyasyonlar DNA erime eğrisi şekilleriyle HRM yönteminde saptanabilmektedir. Kombine yeni nesil DNA boyaları ve geliştirilen gen tarama yazılımları sayesinde güçlü analiz yapabilme kapasitesinin yanı sıra kolay uygulanabilirliği ve düşük maliyetli olma özelliği ile gerçek zamanlı HRM yöntemi pek çok klinik uygulamada ön plana çıkmaktadır.

Anahtar Kelimeler:

Yüksek çözünürlüklü erime, tek nükleotit değişiklikleri, erime eğrisi, mutasyon, metilasyon

High Resolution Melting Method in Molecular Diagnostics and Their Clinical Importance

Along with the development of technology in the field of molecular biology, many new molecular analyses are being developed each day. In this review, we aim to explain high-resolution melting (HRM) analysis, which has been shown to be important in molecular diagnostics, its applications, and its importance in the areas of clinical practice. The HRM system in a closed tube is used to determine post-polymerase chain reaction (PCR)-based genetic variations as a new method. The working principle of HRM is based on the melting behavior of nucleic acid samples. The denaturation of double-stranded DNA is determined by detecting the fluorescence change caused by increased melting temperature. The differences between wild-type and heterozygous samples may be easily detected in melting graphics. Using this method, melting curve analysis can be performed with more precision. In HRM analysis, the samples can be distinguished according to the guanine cytosine (GC) content and sequence length. Thus, the detection of common single-nucleotide polymorphisms (SNPs) in the population, scanning of gene mutations associated with diseases, and analysis of DNA methylation can be performed quickly and reliably using the HRM method. Nucleotide sequence variations and several variations in the PCR products can be detected with the DNA melting curve shape using the HRM method. In addition to being low-cost and easily implementable, the real-time HRM method enables powerful analysis because of the combined new-generation DNA dyes and developed gene scanning software, thereby standing out in many clinical applications.

Keywords:

High-resolution melting, single-nucleotide polymorphism, melting curve, mutation, methylation,

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