Automated Purification of DNA from EDTA-Preserved Whole-Blood Samples: Comparison with Manual DNA Extraction

Year 2008, Volume: 10 Issue: 3, 32 - 38, 01.12.2008

Abdulkadir Yıldırım Hamdullah Turhan Vefa Yanmaz Ebubekir Bakan

Abstract

In recent years, the use of PCR-based molecular techniques in the clinical laboratories has increased. For this purpose, automated DNA isolation instruments using magnetic bead technology have been developed. However, if magnetic particles are not removed sufficiently from DNA solution, amplification reactions will be inhibited by the magnetic beads, which decrease PCR sensitivity or lead to false negative PCR results. The aim of this study was to evaluate PCR-hybridization performance,productivity and purity of genomic DNA extracted by the automated system based on magnetic separation technology, and to compare these results with those obtained with manual DNA isolation method. The yield of genomic DNA extracted by the automated system (QuadroProbe QP10) from human EDTA-preserved whole blood samples was 28 to 74 ng/ l and the A260/A280 ratio was 2.5 ± 0.73. The results obtained from PCR-hybridization showed that the final solutions of DNA isolated by magnetic bead technology did not contain any inhibitory material for the PCR-hybridization reactions. These results show that QuadroProbe QP10 can be used for purification of good quality DNA from whole blood samples in PCR laboratories

Keywords

Automated DNA isolation, magnetic bead, multiplex PCR, hybridization

EDTA'lı Tam Kan örneklerinden Otomatik DNA İzolasyonu: Manuel DNA İzolasyonu ile Karşılaştırma

Abstract

Son zamanlarda klinik laboratuarlarda PCR-tabanlı moleküler tekniklerin kullanımı artmıştır. Bununla birlikte, bu tekniklerden yararlanabilmenin ön şartı, hızlı ve verimli DNA izolasyon yöntemlerine gereksinim duyulmasıdır. Bu amaçla manyetik bilye teknolojisini kullanan otomatik DNA izolasyon sistemleri geliştirilmiştir. Ancak manyetik partiküller elüsyon aşamasında DNA çözeltisinden yeterince uzaklaştırılamazsa, bu manyetik bilyeler amplifikasyon reaksiyonunu bozabilmektedir. Bu durum da PCR sensitivitesini azaltmakta veya yalancı negatif PCR sonuçlarına yol açmaktadır. Bu çalışmanın amacı manyetik bilye teknolojisini kullanan bir otomatik DNA izolasyon cihazıyla elde edilen DNA’ların saflık, verimlilik ve PCR-hibridizasyon performanslarını değerlendirmek ve manuel izolasyon yöntemiyle karşılaştırmaktır. EDTA’lı tam kan örneklerinden otomatik DNA izolasyon sistemiyle (QuadroProbe QP10) elde edilen DNA konsantrasyonları 28 – 74 ng/ l aralığında ve A260/A280 oranı ortalama 2.5 ± 0.73 idi. PCR-hibridizasyon işlemlerinden elde edilen sonuçlar gösterdi ki QuadroProbe QP10 ile izole edilen DNA çözeltisi, PCR-hibridizasyon reaksiyonlarını inhibe edebilecek herhangi bir materyal içermemektedir. PCR laboratuarlarında EDTA’lı tam kan örneklerinden yüksek kalitede DNA pürifikasyonu için QuadroProbe QP10 izolasyon cihazı kullanılabilir

Keywords

Otomatik DNA izolasyonu, manyetik bilye, multipleks PCR, hibridizasyon

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