This module provides an introduction to the practical side of molecular genetics, outlining the major techniques that are used to investigate how genes are expressed and how they are regulated. The general principles of these techniques will be discussed, and some of the tools used by molecular geneticists to perform these techniques will be presented.
Before we look at specific techniques of molecular genetics, we need to understand the general purposes of these techniques. There are many practical applications for these techniques, but they all involve one of just a few general purposes. These basic reasons for using molecular techniques are as follows:
As each technique is discussed, we will look at how it accomplishes one of these purposes.
Formation of Recombinant DNA
Most of the techniques of molecular genetics are either directly or indirectly dependent on molecules discovered in and isolated from bacteria. These molecules have functions in the everyday life of bacteria, but scientists have learned to exploit them as tools used for the manipulation and study of DNA. One vital type of molecule discovered in bacteria is the restriction endonuclease (also known as the restriction enzyme). These enzymes were discovered in 1970 (their discoverers won the Nobel Prize in 1986 for their work), and have since been isolated from hundreds of different species and strains of bacteria, meaning there are hundreds of different restriction enzymes.
So what do these enzymes do? The name endonuclease tells us their function - nuclease is an enzyme that cuts (or cleaves) nucleic acids (such as DNA in this case), and endo means 'within'; therefore, the enzyme cuts within a DNA molecule rather than at the ends. In bacteria, this activity is used to protect bacteria from invasion by bacteriophages - when the bacteriophage injects its DNA, the enzymes cut the DNA and prevent replication of the phage. At least, that's the idea; the strategy isn't always successful.
Restriction enzymes are named according to the species and strain of bacteria they are isolated from. The first letter of the name comes from the genus of the bacterium, the next two letters come from the species, and any subsequent designations are the strain of bacterium and the order in which the enzyme was discovered in that bacterium. For example, the enzyme EcoRI was discovered in Eschericia coli, strain RY13, and it was the first restriction enzyme identified in that bacterium (hence the designation I).
These enzymes recognize specific base sequences within DNA molecules, and cleave specific phosphodiester bonds within that sequence. Different enzymes recognize different sequences, but the sequences recognized share one property - they are all palindromes. A palidrome is a word or phrase that reads backwards the same as it does forwards. In English, this can be a phrase like, "Madam, I'm Adam" or "Sit on a potato pan, Otis". In molecular biology, a palindromic base sequence is one that reads the same in one direction on one strand as it does the other direction on the other strand. (And because DNA strands are antiparallel, that means both sequences are read 5' to 3'.) For example, the palindromic sequence recognized by the restriction enzyme EcoRI is GAATTC. Note (as can be seen in the diagram below) that this sequence (highlighted in red) reads GAATTC on the other strand as well.
When EcoRI encounters its recognized sequence, it cuts the phosphodiester bond between the G and the first A on both sequences (indicated by the green arrows in panel B). This causes the two segments of DNA to separate (as shown in panel C). Note that each fragment of DNA has a four base single-stranded 'tail' protruding from one end. Such a single-stranded projection is often called a "sticky end", which will come into play shortly.
Restriction enzymes are very powerful and useful tools, because they allow researchers to cut large DNA molecules into smaller, more easily manipulated fragments in a very predictable way.
What is the benefit of being able to produce DNA fragments? To fully understand this, we need to introduce the concept of the DNA vector. Vectors are segments of DNA that are replicated in living cells, and have been engineered to allow the insertion of other DNA fragments (like a gene, for example). An example of a vector that we've seen before is a plasmid. There are many types of vectors, including virus DNA, plasmid/virus hybrids, and artificial eukaryotic chromosomes, but plasmids are the most common and easiest to understand. The insertion of DNA into a vector is done by cutting both the vector and DNA of interest (the "insert") with the same restriction enzyme. The sticky ends on each piece of DNA will be complementary to each other, due to the palindromic nature of the restriction enzyme recognition sites. Such DNA Molecules in solution will transiently interact with each other, and join together via hydrogen bonds formed between sticky ends, forming a recombinant DNA moledcule. Four hydrogen bonds are not strong enough to hold these molecules together for more than a few milliseconds, however, and unless the phosphodiester backbones of the DNA molecules are joined together, the two molecules will quickly pull apart. Phosphodiester backbones can by joined together by DNA ligase, which we learned of in the unit on DNA replication. If DNA ligase is present in the mixture with the two different fragments of DNA, any transiently-forming recombinant DNA molecules can be joined permanently to each other. This process is outlined in the diagram below:
Plasmids have several features that allow them to work well as vectors. Some of these features occur naturally in many plasmids whereas others have been engineered into the plasmids to make them more convenient to use. (You don't need to worry about which features are natural and which are engineered.) These features include:
Once created, these recombinant DNA molecules are inserted into bacteria by a process known as transformation, and those bacteria successfully transformed with the DNA can be selected using the selectable marker. These transformed bacteria can then be grown up in great quantities, so that large amounts of the DNA can be recovered. This process, known as DNA cloning, is used for storage and amplification of recombinant DNA molecules. (For more on transformation, see the module on Recombination in Bacteria.)
For more on this process, see the DNA cloning animation on the Russell CD-ROM.