Question

-Describe the process of gel electrophoresis. How would the distance that each DNA fragment travelled on your gel change if you were to make the gel using a higher percentage of agarose (for example 3%)? Explain

-Describe the steps involved in a polymerase chain reaction (PCR), including the changes in temperature that are performed by the thermocycler and the purpose of each temperature change.

Answer 1

Question 1)

Gel electrophoresis is a technique which is used for the separation of DNA fragments, macromolecules and proteins depending on their size and charge. Agarose gel electrophoresis is the method used for the separation of the DNA fragments in the sample based on the size. All the DNA molecules does have same charge per mass, hence the separation of DNA in the gel will be based on their molecular weight. As the name implies, the gel electrophoresis consists of a gel and for the separation of DNA agarose gel is used. Agarose is a polysaccharide and for making the gel the agarose powder is mixed in buffer, heat it to completely dissolve the agarose. After, cooling at room temperature, agarose forms a solid gel in which agarose molecules are held together by hydrogen bonds and the gel will have tiny pores with definite size in it. The samples of DNA are loaded into the wells on one end of the gel and a DNA ladder, a mixture of DNA fragments with known molecular weight will be also loaded into one well. Then an electric current is applied in which the negative electrode will be at the side of sample wells and the positive electrode will be in the other end. Because of the negative charge of the DNA, the DNA molecule travel from the negative charge to towards the positive charge. Based on the size of the DNA fragments in the sample, the molecules moves differently through the gel. The shorter DNA fragments moves faster through the pores where as the larger DNA fragments move slowly due to the the steric hindrance. Once the DNA fragments are separated, the gel is subjected to stain with DNA binding dye like ethidium bromide. The stained gel under the UV light, the DNA bands will glow which enables us to locate the position of the DNA fragments in the gel and with respect to the DNA ladder, the size of the DNA fragments in the sample is determined.

The concentration of the agarose and the pore size of the gel is inversely proportional. As the concentration of agarose increases, the pore size decreases. As a result, only shorter DNA fragments can travel through the pores in the gel and the bigger ones will remain in the gel. A lower concentration of agar enhances the separation of higher molecular weigh DNA fragments in the sample whereas, the higher concentration of agarose gives good resolution for smaller fragments. The standard percentage of agarose used for DNA separation is 1%.

Question 2)

Polymerase chain reaction or PCR is molecular biology technique used for amplifying a specific DNA sequence. PCR provided millions of copies of a segment of DNA of interest. Thermo cycler is the instrument used for carrying out PCR. The main steps involve in PCR is given below.

1. Denaturation:

In this step the two strands of the DNA molecule is separated. The separation of DNA strand is achieved by increasing the temperature leading to the breaking of the hydrogen bonds between the complementary DNA strands. In this the temperature of the reaction chamber is increased to 94–96 °C for around 20–30 seconds as a result the double stranded DNA is melted producing two single strands.

2. Annealing:

In this step, the primers which are specific to the DNA sequence of interest binds to the targeted site on the DNA strands. One primer binds with only one strand of the DNA at their 3' end. During the annealing process the temperature of the thermo cycler will be lowered to 50–65 °C for 20–40 seconds. The annealing temperature is determined based on the primers used. The specificity and the efficiency of the primer binding to the targeted DNA strand depends on the annealing temperature. If the temperature is too less, there is chances that the primer binds imperfectly with the DNA strand and if the temperature is very high the primer may not be able to bind at all.

3. Extension:

During extension, the enzyme DNA polymerase synthesize the complementary sequence of both the strands by extending the primer. The enzyme add corresponding nucleotides to the free 3' OH end of the primer and synthesize the complete strand. The temperature in the reaction chamber of the thermo cycler will be 72°C during extension because it is the optimum temperature for the enzyme activity of DNA polymerase enzyme.

The figure below shows, the steps involved in PCR: