DNA Sequencing

Ultimately, to properly study and utilize a piece of DNA, we need to know its sequence of bases. This can be done using a technique called DNA sequencing, which utilizes a modified DNA replication reaction. As with other DNA replication reactions, this one requires a primer. For DNA sequencing, the primer is often complementary to a sequence within the vector, close to where the DNA of interest is inserted. To visualize the synthesized DNA, the primer is labeled, either radioactively or with a fluorescent tag. Other components of the reaction are normal components of a DNA replication reaction, except for one. One of the nucleotides is a dideoxynucleotide triphosphate (ddNTP). This means that in addition to lacking a hydroxyl group at the 2' position of the sugar, it also lacks a hydroxyl group at the 3' position of the sugar. Think about what this would mean to DNA replication. Where does DNA polymerase add a free nucleotide to on the growing polynucleotide chain? That's right - to the 3' hydroxyl of the nucleotide at the end of the chain. If the nucleotide at the end of the chain has no 3' hydroxyl (if it is a ddNTP), then there is no place for DNA polymerase to add another nucleotide and DNA synthesis will stop.

So how does this help determine the sequence of the bases? Imagine the following reaction: a standard DNA replication reaction with a template (the DNA being sequenced), a primer (labeled), DNA polymerase, all four dNTPs, and one ddNTP (for example, let's say it is ddATP) at a low concentration. Because the ddNTP is at a low concentration, it will be inserted into the DNA chain only occasionally and randomly - sometimes in the first A position, sometimes in the second A position (with a normal A in the first position), sometimes in the third A position, etc. The result would be a mixture of labeled DNA molecules, all terminating at different positions, but all ending with A. If this reaction is run on a gel, it will produce a ladder of bands. Now extend this by adding three more separate reactions, each with a different ddNTP. There will be one reaction with fragments all ending in C, one with fragments all ending in G, and one with fragments all ending in T. Each of these is run on an adjacent lane in the gel, and the sequence can be read right off the gel, because longer fragments will not run as far as smaller fragments, so the fragments can be ordered in terms of size, and we know the base that ends each fragment.

For a nice presentation of the process of DNA sequencing, check out the DNA sequencing animation on the Russell CD-ROM.

Expression of Cloned Genes

Ultimately, the main reason we clone and characterize genes is so we can produce the protein. This is done for a variety of reasons. Sometimes, the protein is a product that will be harvested for commercial sale (for example, by a biotechnology or pharmaceutical company). Production of the protein can also be used in an indirect way to understand its function. By expressing the gene in cells or an organism (for example, expressing a gene at a time or in a place that it wouldn't normally be expressed), it may be possible to observe the effect that expression has on the organism, and infer the function from that. So how do you express a gene? There is a class of vector known as an expression vector. These vectors have promoters (often from viral genes) engineered into the vector near the multiple cloning site. Any gene inserted into the multiple cloning site therefore comes under the control of the promoter, allowing expression of the gene under appropriate conditions. Expression may be in vivo (in a living cell or organism) or in vitro (in a test tube using cell-free transcription and translation systems that are available commercially, allowing production of the protein relatively purely).

Techniques of Molecular Genetics: Summary of Key Points