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 an 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 will be provided with a sample containing a plasmid vector. This vector has had a gene for a short region of the protein PLK1 inserted into it through ligation. Due to the nature of DNA, that gene may have been inserted correctly or back-to-front. Using a small sample of the plasmid, you will perform a restriction digest to “drop out” the insert and use electrophoresis to check its orientation.

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

Using plasmids for cloning

One of the more common techniques available to scientists working in molecular biology is cloning. In this technique, sequences of DNA containing genes of interest are inserted into vectors which are then used to introduce these genes into cells or organisms to study the effects of the expression of the genes.

Vectors are based on bacterial plasmids – short circular pieces of DNA separate to the main bacterial chromosome which may be transferred between bacteria. Scientists source plasmid vectors from biological supply companies, which create them by ligating together pre-existing genes and sequences of DNA built from scratch using sequencing technology. An example of a commercial vector is the pGEM-T Easy system.

Prior to today’s project, a gene for a fragment of a protein called pololike kinase 1 (PLK1) has been inserted into a pGEM-T Easy vector by another group of students. The Diamantina Institute’s Cell Cycle research group hopes to use these vectors to study where PLK1 localises in the cell during the cell cycle in order to find out how changes to the cell cycle might lead to cancer.

DNA is a symmetrical molecule, meaning that during the process of transcription, either strand could act as a template for the creation of the mRNA molecule. As each strand runs in an opposite direction to the other, a gene could in theory be transcribed forwards or backwards. In the cell nucleus, genes are only transcribed in one direction due to the placement of promoter sequences upstream of the gene. In our vector, however, the promoters are found on the vector itself rather than the insert. Therefore there may be some of the samples where the vector has been inserted the right way around, and others where it has been inserted backwards. We only want the samples where the vector has been inserted the right way around. Luckily, we can use a restriction digest to find out this information

Restriction digests to check orientation

Restriction enzymes cut the DNA strand at very specific locations, normally given by sequences of half a dozen or so base pairs. The pGEM-T Easy vector has been created with a number of restriction sites on either side of the insertion point. Some of these restriction sites are only found on one side of the insertion point, and so can be used to linearise the vector to make it ready for electrophoresis. Others are found on both sides, and so may be used to “drop out” the insert to check its size.

Electrophoresis separates fragments of DNA into its component sizes. Fragments of the same size travel through the gel in one region, resulting in a “band” of DNA. When we compare the band to bands of known size, we can estimate the size of the fragments in our band. Consider the following example :

The enzyme EcoRI has restriction sites on both sides of the insertion point. This means that if we expose our sample to EcoRI, we should end up with two sorts of fragment – one representing the vector (around 3000 bases long, or 3kb) and one representing our insert, which in this case is 700 bases long (0.7kb). This will appear on the gel as indicated in Figure 1.

EcoRI digest of Polobox insert in pGEM-T Easy

This technique allows us to check whether the insert has gone into the vector (otherwise you would just see a band at 3kb) for the vector. However, it does not tell us which way around the insert is oriented. To do this we need to use a different restriction enzyme.

SalI has a single cutting point just downstream of the insertion point in the vector. Normally, a digest using this enzyme would result in a linearised plasmid and a single band at 3.7kb (3kb vector + 0.7kb insert). As luck would have it, however, the inserted gene also contains a SalI cutting point close to the 5’ end. This means that there are two possibilities, depending on how the insert is oriented (see Figure 2).

SAlI digests of Polobox inserts in pGEM-T Easy in forward and reverse orientation

In the incorrect orientation example above, the SalI sites are so close together that the fragment dropped out will be too small to resolve using this gel.