Team:HK SKHLPSS/Protocols
Protocols
In iGEM 2017, a DNA three dimensional nano-structure was successfully designed to detect the presence and concentration of H3N2 influenza mRNA biomarker. However, the H3N2 influenza biomarker was synthesized in commercial lab. Designed DNA nano-structure is therefore detecting mRNA biomarker theoretically.
Assembly of DNA nano-structure
DNA sequence of Lactobacillus was found on Basic Local Alignment Search Tool (BLAST) from National Center for Biotechnology Information, U.S . Several nucleotide bases were selected as the target DNA sequence of Lactobacillus and used as the design of nano-device later on.
Four single-stranded DNAs bought from IDT Technologies were mixed and placed in the Dynamica C-Master Gradient thermal cycler. Starting at 95oC, the temperature was then dropped 1oC per minute until room temperature was achieved. The drop of temperature allowed four single stranded DNAs to anneal according to our design by complementary base pairing principle, forming the nano-structure.
To prove the DNA nano-structure was actually formed in accordance with our design, 125 Polyacrylamide gel electrophoresis (PAGE) was conducted. Four distinct single stranded DNAs and target DNA (10μL, 150nM) were loaded separately. Different combination of four single stranded DNAs were mixed and placed in thermal cycler before loaded into the gel to check whether each strand were bound as we expected. Lastly, the nano-structure was mixed with the target DNA before running the gel to check whether the nano-structure was bound with Lactobacillus target DNA. Constant voltage at 80V was maintained with Gelred used during PAGE. Detailed loading combination in each well could be found in the result part of this wiki.
Peroxidase assay for target DNA fragment
Peroxidase assay was conducted for two reasons. Firstly, it was used to prove the nano-structure was actually formed. Secondly, it was used to check for whether the nano-structure could actually reflect the concentration of Lactobacillus in samples.
In this respect, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) peroxidase assay was employed to check for the peroxidase activity of G-quadruplex inside the nano-structure. In our assay, target DNA fragment (synthesized in IDT Technologies) and nano-structure (100 nM final), and hemin (63μM) are added to buffer .The mixture was then incubated at room temperature for 30 minutes in a shaker (60rpm). 15μL ABTS solution (20mM final) and H2O2 (20mM final) were added to the mixture. The reaction mixture was then transferred to a 96-well plate and absorbance at 405 nm was measured and recorded with a microplate spectrophotometer.
DNA extraction of Lactobacillus followed by peroxidase assay
Raw sample was extracted from commercial products. Qiagen DNeasy Blood & Tissue Kit was used to extract Lactobacillus DNA from the raw sample. After that, the same peroxidase assay was conducted except Lactobacillus DNA fragment was replaced by the extracted sample from the commercial product.
