The project

Many molecular biology techniques take a significant amount of time to complete. Many of the enzyme-based reactions which underpin these techniques require incubation periods of and hour or more, while cloning and transformation often requires overnight incubation to allow the transformed cells time to recover and multiply. In the limited time we have available, we cannot hope to cover all molecular biology techniques, however you will undertake a mini-project which will expose you to some of the more important DNA-based techniques.

In this project you are tasked with generating multiple copies of a region of a gene using the polymerase chain reaction (PCR). Once the reaction is complete, you will demonstrate the presence of the DNA fragment using agarose gel electrophoresis.

Getting started

Before you begin, make sure that you are familiar with the relevant theory behind the techniques we will be performing. This manual contains several appendices which will provide you with this information. Make sure you read this information before proceeding.

Other molecular biology techniques are provided at the SPARQ-ed website here.

Theoretical basis of the project

The Polymerase Chain Reaction

Scientists have investigated the nature and role of DNA ever since Watson and Crick reported its structure in 1953. The vital role that this molecule plays in all living organisms means that it can be used in a wide variety of applications, from identification of an individual due to their unique DNA profile, to engineering living things so that they are better suited to survival, or can be used to produce useful products.

Until recently, however, this application of our knowledge of DNA was limited by the amount of DNA we could get our hands on. If we wanted to introduce a gene for a useful protein into another organism, we had to recover that gene initially from large quantities of tissue from the organism where the gene was found. If we wanted to use DNA profiling to identify a suspect in a criminal case, we had to have large quantities of tissue or body fluids form the suspect to be able to demonstrate the profile. This changed with the development of the polymerase chain reaction, or PCR.

In 1983, Kary Mullis reported a technique which utilized DNA’s ability to self-replicate in the presence of enzymes called polymerases and the nucleotides which form the building blocks of DNA. Using polymerases from thermophillic (“heat-loving”) bacteria which operate at temperatures higher than most organisms can tolerate and custom made primers composed of single strands of DNA complementary to regions around the gene of interest, Mullis’ new technique allowed scientists to make billions of copies of a single length of DNA. Now, large quantities of DNA for genetic transformation can be synthesized at a relatively low cost, and enough DNA to produce a profile could be derived from a single hair or drop of blood.

PCR consists of three main stages :

  • Denaturation – heating the DNA to around 95°C to separate the two strands of DNA
  • Annealing – cooling the DNA to 50-60°C to allow the primers to attach, and
  • Extension – heating the DNA to 72°C so that the polymerase enzymes can complete the complementary strands from the nucleotides, starting at the primers

This process is repeated 25-35 times, with the DNA roughly doubling every cycle. When Mullis first developed the procedure he performed each cycle manually using heated waterbaths, however the mechanical nature of the process made it well suited to automation, and most PCR is now performed using PCR cyclers which can be programmed to perform reactions using different combinations of time and temperature. PCR now forms the basis of many molecular biology investigations. For his contribution to science, Kary Mullis was awarded the Nobel prize for chemistry in 1993. A more detailed explanation of PCR can be found here.

In this exercise, you will be amplifying a small region of the gene for a protein called polo-like kinase 1 (PLK1), which is involved in the regulation of the cell cycle and plays an important role in cellular processes which occur during mitosis. The region of the PLK1 gene we will be amplifying codes for a part of the protein called the polobox domain. This domain allows PLK1 to localize to various parts of the cell during the cell cycle. A knowledge of this part of the gene is useful, as disturbances to the normal function and localization of PLK1 is found in some conditions caused by changes to the normal cell cycle, such as cancer

One way of thinking about what you are doing in this exercise is to imagine the human genome as an enormous library with 23,000 books, each book representing a gene for a particular protein. We have provided you with a copy of the “PLK1 book” (the template DNA, a circular length of DNA called a plasmid which contains the gene for PLK1). Your task will be the equivalent of making billions of copies of the “polobox page” of the PLK1 book. To help you with this, we have also given you “bookmarks” in the form of primers which are upstream (before) and downstream (after) the polobox page and plenty of “copier paper” (nucleotides). You will use the molecular biology equivalent of a photocopier (Pfu polymerase and a PCR cycler) to make these copies.

Agarose gel electrophoresis

Most of this procedure involves adding minute quantities (eg. 1μL, the volume enclosed by a cube 1mm on a side) of colourless liquid to minute quantities of other colourless liquids. When the reaction is complete, you are left with a small volume of colourless liquid which looks no different to what you started with. Therefore you will need to use a technique which shows you have made the correct DNA. This is where electrophoresis comes in.

If we wanted to sort sand from gravel from larger rocks, we would use a series of sieves of different sizes. Each sized sieve lets smaller particles pass through but retains the larger fragments. Electrophoresis can be thought of as a sieve for large molecules like DNA or protein.

In agarose gel electrophoresis, a DNA sample is loaded towards one end of a block of a jelly-like substance called agarose. When an electrical current is passed through the gel, the DNA molecules are pushed through the gel away from the negative electrode (DNA has an overall negative charge and like charges repel). Smaller fragments of DNA can move more easily through the gel than larger fragments, so in a given period of time, DNA of different sizes accumulates in regions of the gel. If we include a dye which binds to the DNA, these regions are visible as bands – the further towards the positive electrode a band is located, the smaller the fragments of DNA are found in that band.

To get an idea of the size of a band seen on a gel, we always run a sample consisting of a mixture of DNA fragments of known sizes alongside our test samples. This is called a marker, or a “ladder”, as the multiple bands of DNA seen on the gel resembles the rungs on a ladder. By matching the position of a band in our test sample to those representing DNA of known size in the ladder, we can estimate the size of DNA fragments in our test. The part of the PLK1 gene which codes for the polobox domain is 800 base pairs (bp) long. Therefore, if our PCR has been a success, we should see a band corresponding to our DNA markers which are 800 base pairs long.

More detailed information on electrophoresis can be found here.