IGEM:IMPERIAL/2007/Tutorials/Guide for Engineers/Standard Lab Techniques
- 1 Introduction
- 2 The Molecular Biologist Toolbox Kit
- 3 Gene Cloning
- 4 Gel Electrophoresis
- 5 Blotting Techniques
- 6 PCR (Polymerase Chain Reaction)
- 7 DNA Sequencing
The Molecular Biologist Toolbox Kit
Restriction enzymes are a class of enzyme that can causes specific breaks in a DNA molecule.The mechanism by which restriction enzymes cut DNA is by causing hydrolysis of the phosphodiester bonds between the nucleotides that make up DNA.
Restriction enzymes can be broadly split into two types:
- Exonucleases cleave DNA from the ends of the DNA molecule. The action of exonucleases will remove nucleotides from the ends of DNA.
- Endonucleases cleave within the interior of the DNA molecule. The action of endonucleases will cause fragmentation of DNA molecules.
Endonucleases are essential for recombinant DNA technology because they allow specific genes to be cut out and released from within a DNA molecule. The specificity of endonucleases is determined by a specific sequence of nucleotides that they can recognise and cleave.
This sequence of nucleotides is called the restriction site and will vary between different types of endonucleases.
The key features of the restriction sites are the sequence of nucleotides, sequence length and site of cleavage. In addition most of the restriction sites are palidromic, meaning that they read the same forwards and backwards. If this is the case then there is cleavage of both strands of DNA that make up the DNA molecule.
Once a DNA molecule has been cleaved two fragments will be resolved. Each of these fragments will contain an 'end' that is from the cleaved restriction site. These ends can vary, depending on the exact position of the bonds cleaved. In general there are two broad types of ends resolved from fragmentation:
- Blunt ends where the two strands of DNA are the same length, and so there is no overhanging strand. This types of ends occur when the cleavage sites on both strands are directly opposite each other.
- Sticky ends where one of the stands of DNA is longer and so overhangs the other. This type of end occurs when the cleavage sites on both strands are not opposite each other. The examples shown in the diagram produce this types of end.
Vectors and Plasmids
Gel electrophoresis is a technique used to separate biological molecules such as RNA, DNA and proteins through a gel support. The gels are a porous network of cross-linked polymers, usually composed of agarose or polyacrylamide. The principle of separation is to apply an electric field to drive charged molecules through the gel and separated out a complex mixture into distinct bands. The separation is based upon the mobility of molecules through the gel.
The mobility of molecules can be defined as:
A key use of gel electrophoresis is to separate mixture of DNA molecules. DNA is negatively charged and so, when an electric field is applied the DNA molecules will move towards the positive node.
Generally speaking the key factor determining the mobility of DNA is the length of the molecule(molecular dimension). The reason why charge is not a factor for DNA mobility, is that DNA of all lengths have the same mass to charge ratio. In addition other factors affecting mobility include conditions of electrophoresis and conformation. The conformation is the form that the DNA is in i.e. in a linear, circular or supercoiled form.
As the size of DNA molecules increases the distance migrated decreases, this is because larger strands of DNA will have a greater steric resistance in the gel and so migrate slower. In addition for linear DNA, the distance migrated shows a linear correlation to the length of DNA. This correlation enables a DNA marker to be run out and a graph of distance migrated vs length of DNA plotted. Using this a band of unknown DNA size can have its distance migrated measured and the approximate length worked out.