Optimization of High Concentration Plasmid DNA for Use in COVID-19 mRNA Vaccine Development: Comparison of Between Alkaline Lysis Method and Commercial Kit Results

Year 2022, , 154 - 161, 30.12.2022

Tuğçe Duran , Naci Çine , Nadir Koçak , Saliha Kurt

https://doi.org/10.31196/huvfd.1151456

Abstract

While forming the stable IVT mRNA molecule, high concentration and purity plasmid DNA must be obtained to ligase the ORF antigen sequence initially copied from the plasmid DNA with the UTR regions. In this study, in the stage of creating the mRNA molecule, which is the first step of the COVID-19 mRNA vaccine, comparison and optimization of the pDNA containing the ORF target antigen sequence were performed as a result of isolation with alkaline lysis method and commercial kit. Plasmid DNA bacteria containing the target antigen ORF sequence were grown under appropriate conditions. Plasmid DNA was isolated by commercial kit and alkaline lysis method from bacterial cultures stopped at different OD600 nm values (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5). After the obtained pDNAs were visualized on an agarose gel, their purity and concentration were measured by spectroscopic measurement. After the stab culture is resuscitated in SOC medium, bands are formed in a single form after isolation with the kit, and in multiple forms (linear, supercoiled, circular) after pDNA isolation by alkaline lysis method. The ideal OD600 nm for both methods was 0.3-0.4. As a result of isolation with the kit, higher purity on the contrary low concentration pDNA was obtained. The ideal OD600 nm value is a critical parameter that affects the concentration and purity of pDNA. The alkaline lysis method is a cheap and powerful technique that can be used as an alternative for mRNA vaccine development compared to kit isolation.

Keywords

Plasmid DNA, isolation, alkalen lysis, kit, mRNA vaccine

Supporting Institution

Kocaeli University Scientific Research Projects Coordination Office

Project Number

2021/2663

COVID-19 mRNA Aşı Geliştirilmesinde Kullanılacak Olan Yüksek Konsantrasyondaki Plazmid DNA’sının Optimizasyonu: Alkalen Lizis Metodu ve Ticari Kit Sonuçlarının Kıyaslanması

Abstract

Kararlı IVT mRNA molekülü oluşturulurken başlangıçta plazmid DNA’sından kopyalanan ORF antijen sekansının UTR bölgeleri ile ligaze edilebilmesi için yüksek konsantrasyon ve saflıkta plazmid DNA'nın elde edilmesi gerekmektedir. Bu çalışmada, COVID-19 mRNA aşısının ilk basamağı olan mRNA molekülünün oluşturulması adımında, ORF hedef antijen sekansını içeren pDNA’nın alkalen lizis method ve ticari kit ile izolasyonu sonucundaki kıyaslama ve optimizasyonu yapılmıştır. Hedef antijen ORF sekansını içeren plazmid DNA bakterisi uygun üreme koşullarında çoğaltılmıştır. Farklı OD600 nm değerlerinde (0.02-0.05, 0.05-0.1, 0.1-0.2, 0.2-0.3, 0.3-0.4, 0.4-0.5) durdurulan bakteri kültürlerinden ticari kit ve alkalen lizis yöntemi ile plazmid DNA izolasyonu yapılmıştır. Elde edilen pDNA’lar bir agaroz jelde görüntülendikten sonra, spektroskopik ölçüme tabi tutularak saflığı ve konsantrasyonu ölçülmüştür. Stab kültür SOC medium içerisinde canlandırıldıktan sonra, kit ile izolasyon sonrasında tek formda, alkalen lizis metodu ile pDNA izolasyonu sonrasında birden çok formda (lineer, süper kıvrımlı, sirküler) bant oluşur. Her iki yöntem için de ideal OD600 nm 0.3-0.4 olarak bulunmuştur. Kit ile izolasyon sonucunda daha yüksek saflıkta ancak düşük konsantrasyonda pDNA elde edilmiştir. İdeal OD600 nm değeri, pDNA'nın konsantrasyonunu ve saflığını etkileyen önemli bir parametredir. Alkalen lizis yöntemi, kit izolasyonuna kıyasla mRNA aşı geliştirmeye alternatif olarak kullanılabilecek ucuz ve güçlü bir tekniktir.

Keywords

Plazmid DNA, izolasyon, alkalen lizis, kit, mRNA aşısı

Project Number

2021/2663

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