Restriction Endonuclease Digestion of Plasmid Dna
Introduction: With the execution of this experiment, we began to go deeper into the Cell and Molecular Biology course. The main focus of the experiment would be how the Restriction Endonucleases cleave the strands of DNA. For this experiment, pBR322 was the specimen to use. Restriction Endonucleases work by cleaving the sugar phosphate backbone of specific DNA sites. Restriction enzymes that have been isolated from bacteria have a defensive role. This idea is illustrated when an attacking foreign cell DNA is trying to alter the bacteria; restriction enzymes cleave the DNA rendering it inert.
The second part of the experiment deals with Gel Electrophoresis. The samples are loaded into wells on an 1% agarose slab and subjected to electrical currents both positive and negative. Our current target here is DNA, therefore since nucleic acid as a negative charge, the bands will migrate toward the positive cathode. This process of migration is called sieving and smaller strands move faster than longer strands due to their ease in going through the gel. The objectives of the experiment include:
Learning the principles behind Restriction Enzymes and Gel Electrophoresis Applying the concepts in the experiment to produce bands at the end of the Gel Electrophoresis stage Interpreting what these bands mean with accordance to how the plasmid was cleaved Methods and Materials: For the experiment we used several restriction endonucleases (BamHI, EcoRI, HindIII, PstI, ScaI, SaII), ppBR322 plasmid DNA, TAE/TE Buffer, DNA Ladder (50 Bp), Restriction Buffers, 1g of Agarose, 700ml of Distilled H2O. Equipment used for the experiment included: Agarose Gel Electrophoresis System, Uv-vis illuminator and Camera or a Gel doc-it documentation system.
The first procedure began by adding 8. 5 µL sterile distilled H2O, 1. 0µL of the appropriate 10x buffer, 1. 0µL combination of the restriction endonucleases and 1. 0µL of pBR322 plasmid DNA (the DNA would be added last) in 5 separate 1. 5ml microcentrifuge tubes, one tube is not to have an RE in it. The mixture was then incubated for 1 hour at 37 °C. No dry block heater was available so body heat was used. After incubation, 2µL of gel loading dye (Bromphenol Blue) was added to each mixture and loaded in 1% agarose gel. The 50bp DNA ladder was placed in lane 1.
It was then subjected to electrophoresis at 100V 250mA 50W. Agarose gel was prepared by dissolving 1g of agarose gel powder in 100mL distilled H2O in a microwave over. It was then cooled at 60°C then poured in a gel casting tray. A comb was then put and the gel was left to solidify. Afterwards, the gel casting tray was placed into the submarine gel electrophoresis system. The TAE buffer was then placed. The samples were then loaded from left to right starting with the DNA ladder on lane with and the sample without any restriction enzyme on the extreme right.
It was then covered and the anodes were connected on the side of the walls. They were connected to the power supply set at 100V 250 mA 50W and then run. When the tracking dye reached near the end point, the power supply was turned off. The gel was then removed and transferred into a developing try containing a 10µL ethidium bromide pero 100ml buffer. It was then shook for 15 minutes. The get was then transferred to the documentation system and Rf values were measured. Pictures were taken and the gel was immersed in hypocholorite (chlorox) solution before discarding. Results and Discussion
The group did not include a mix without restriction enzymes because doing so will lead to undigested or incompletely digested DNA. The DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor. In prokaryotes, the major role of DNA methylation is to protect host DNA against degradation by restriction enzymes. In eukaryotes, DNA methylation has been implicated in the control of several cellular processes, including ifferentiation, gene regulation, and embryonic development. Structural work on HhaI DNA methyltransferase demonstrates that the substrate nucleotide is completely flipped out of the helix during the modification reaction and has provided much insight into the enzymatic properties of S-adenosyl-L-methionine (SAM)-dependent DNA-modifying enzymes. Structural comparison of three enzymes, HhaI C5-cytosine methyltransferase, TaqI N6-adenine methyltransferase, and catechol O-methyltransferase, reveals a striking similarity in protein folding and indicates that many SAM-dependent methyltransferases have a common catalytic-domain structure.
This feature permits the prediction of tertiary structure for other DNA, RNA, protein, and small-molecule methyltransferases from their amino acid sequences, including the eukaryotic CpG methyltransferases. Ethidium bromide is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology laboratories for techniques such as agarose gel electrophoresis. It is commonly abbreviated as “EtBr”, which is also an abbreviation for bromoethane.
When exposed to ultraviolet light, it will fluoresce with an orange colour, intensifying almost 20-fold after binding to DNA. Ethidium bromide is an “intercalating dye”, that is, it is able to slip itself into the DNA while essentially stacking itself between the bases of the helix. When it is inserted into the DNA, it becomes much more fluorescent when exposed to ultraviolet light as compared to ethidium bromide just in solution. So we can use it to visualize the DNA that has been resolved on a gel by electrophoresis.