Question

Briefly describe the process of electrophoresis. Be sure to address the polarity of DNA, the movement of DNA through the agarose gel, and the construction of the gel elec- trophoresis chamber. You may draw and label a figure if you wish. 12 Why is electrophoresis a useful tool in modern DNA analysis? What would be some practical applications of this technique?

Answer 1

Ans 1) Process of electrophoresis helps in tracking the movement of the molecules in a gel under the influence of electric field. The molecule move in the electrophoresis, the movement of the molecules depends on the charge of the ions. The technique is used in the labs to separate the molecules based on their size and hence in this electric field with negative charge is applied and hence the molecules move towards the positive charge. It helps in analyzing as well as separating the molecules like RNA, DNA, proteins, nucleic acid etc. There are two factors which helps in tracking as well as controlling the movement of the particles which includes the charge of the same. The species which are negatively charged moves to the positive side of the electric field and the species which are positively charged moves towards the negative end. The second factor which determines electrophoresis is the size of the molecule where the smaller ones moves much quicker than the larger ones.

The DNA molecules are negatively charged due to the presence of the phosphate bond and hence when the separation is obtained on the electrophoresis, the DNA molecules on application of electric field moves towards the positive pole. Hence when the electric field is applied the DNA moves towards the anode within the electrophoretic gel. When it comes to the polarity of the DNA, one end has phosphate towards the 5’-end of the carbon. Due to the higher number of phosphate group, the DNA backbone carry more negative charge.

Preparation of electrophoresis

• The first step is the DNA is isolated and processed so that it can be used to run on the gel solution. The DNA solution is mixed with some basic dye so that its movement can be tracked and visualized along the gel.
• This is followed by the preparation of the TAE buffer which is very important as it helps in creating the electric field in the chamber. The percentage of agarose is determined depending on the size of the band expected from the DBA. When a smaller band is expected, a higher percentage of agarose gel is to be used and when larger band is expected, less amount of agarose gel is used.
• After this the casting tray is used in which all the sides are closed with cello tape, gel is poured and allowed to solidify.
• The electrophoresis chamber is set up after that where the chamber is filled with TAE buffer and gel tray is position close to the negative electrode.
• After the gel wells are loaded with the sample of the DNA and a ladder DNA is used to make the comparison with rest of the size.
• After this the negative and positive leads of the chamber are connected with power supply and the voltage is turned on. As the voltage is turned on, it initiates or starts the electric field and hence the DNA which is negatively charged starts to migrate towards the positive electrode.

Ans 2) Gel electrophoresis is an important analytical tool because it helps in separating the molecules based on their size as well as their charge. It helps in estimating the size of the DNA molecules as well as it helps in analyzing the PCR products. It has many application in different field of subjects like forensics, genetics, microbiology, molecule biology etc.

Applications of the electrophoresis are:-

• It helps in DNA analysis where it studies and identify the DNA fragments as they move along the electric field. The larger and smaller fragments of DNA are able to separate differently and hence it is an important tool for comparing DNA especially in forensic studies.
• It helps in identifying certain behavior of proteins and hence helps in identifying many disease and health conditions. It can be performed to identify the urine and blood sample and hence through interaction with certain antibodies, it helps in quantifying the proteins.
• It helps in testing the antibiotic purity as well as it helps in testing wide range of vaccine.