Folding of the Vector (pCDH-CMV-MCS-EF1-puro) into Predefined Shape Using 20mer Staples
The folding of DNA to create nanoscale shapes is known more commonly as DNA origami. DNA origami is a fairly new technique in field of nanobiotechnology. It is mainly used in drug target transfer process and drug delivery. It is also used in making fairly simple designs such as smileys, dolphins, trapezium square etc. This again used to create three dimensional structures too for example cube, capsules etc. The present project aims to fold the DNA in the shape of a bell.Double stranded DNA (pCDH–CMV–MCS–EF1–puro, 7.384Kb size) was used as a scaffold which is isolated from DH5- α E coli clone. Miniprep and Maxiprep were performed in order prepare sufficient amount of scaffold (Ref). Agarose Gel Electrophoresis and Spectrophotometric analysis (EppendorfBiophotometer). The circularly permuted plasmid was restricted by unique enzymes- Kpn I, Xba I and Not I to generate a linear plasmid. The band representing the linear plasmid was gel eluted using the Qiagen gel elution kit. Eighteeen, 20mer staples were designed and custom made from Sigma which are unique in its sequence and produced no dimmers with any of the other staples. Folding was performed when 10nM of scaffold strands were mixed with 50nM oligonucleotide staple (1:5 ratio) strands by carrying out a single pot PCR reaction. The formation of the structure was visualised through Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM).