Question

From your laboratory data, you were able to estimate the approximate size of each of the DNA fragments that you separated on your gel. This was done in terms of the number of base pairs. Explain how you made this determination.

Answer 1

Answer: The DNA fragments are separated in an electrophoresis gel and compared with a  weight marker, a reference standard containing DNA fragments of known lengths

Explanation:

Gel electrophoresis is a lab technique used to separate DNA according to their size. However, the DNA molecules in the cells are too large to separate through a normal electrophoresis gel, but they can be analyzed if they have previously been fragmented, for example, using restriction enzymes.

Agarose gels (concentration between 0.3% and 2%) are usually used to separate DNA, because they are more porous than polyacrylamide gels.

First, the gel is placed in a chamber with a buffer that allows the conduction of an electric current. One end of that chamber is connected to a negative electrode, while the other end is connected to a positive electrode. So, DNA samples are loaded into a slot next to the negative electrode and an electric current is applied that makes them move through the gel. And, one well is used for a reference standard which has DNA fragments of known lengths. Commercial DNA markers cover different size ranges, so it is important to choose one with good "coverage" in the size range in which we expect to find our fragments. Since the DNA fragments have a negative charge, they will move towards the positive electrode. Thereby, small fragments move through the gel faster than large ones.

At the end, longer fragments will stay close to the negative end as being larger, they move more slowly. And shorter fragments will be closer to the positive end of the gel, because they will move faster.

The next step is to stain the gel with a pigment that binds to the DNA, and the fragments can be seen as bands under UV light allowing us to see the DNA present at different locations along the gel. It should be noted that a single DNA fragment would not be visible. So actually. each band contains a great number of DNA fragments of the same size at the same position.

By comparing a band in a sample with the molecular weight marker, we can determine its approximate size. However, in order to be more precise, we can draw a calibration curve. You can measure the advanced of the electrophoresis front versus the logarithm of the size (lbase pairs) for each band and calculate a regression line. So the advance distances of the samples are interpolated, which will allow you to to calculate the of a specific fragment.