Recombinant DNA and genetic techniques for higher education
Recombinant DNA (or rDNA) is made by combining DNA from two or more sources. In practice, the process often involves combining the DNA of different organisms. The process depends on the ability of cut, and re-join, DNA molecules at points identified by specific sequences of nucleotide bases called restriction sites. DNA fragments are cut out of their normal position in the chromosome using restriction enzymes (also called restriction endonucleases) and then inserted into other chromosomes or DNA molecules using enzymes called ligases.
A fragment of DNA, containing a single gene or a number of genes, can be inserted into a vector that can be propagated within another cell. A vector is a section of DNA that can incorporate another DNA fragment without losing the capacity for self-replication, and a vector containing an additional DNA fragment is known as a hybrid vector. If the fragment of DNA includes one or more genes the process is referred to as gene cloning.
- Plasmid vectors are modified forms of the circular extra-chromosomal DNA molecules found in bacteria, which have been engineered to contain restriction sites and marker genes (to allow the detection of bacterial cells that contain the plasmid). The bacterial artificial chromosome (BAC), a vector based on the naturally occurring F-plasmid found in the bacterium Escherichia coli, is used to clone relatively large segments of DNA.
- Lamda phage vectors are recombinant viruses, containing the phage chromosome plus inserted 'foreign' DNA. In general, phage vectors can carry larger DNA sequences than plasmid vectors.
- Cosmids are artificially constructed cloning vectors, produced by combining plasmids with sections of the lambda phage chromosome. The resultant cosmid can be packed into the phage body for transmission, and use the plasmid genes to direct its replication within the host cell.
- Expression vectors are vectors - plasmid or phage - that include regulatory sequences necessary for the transcription and translation of the cloned gene. The aim is to make as many copies of the protein coded by the gene as possible within the host cell.
The host cell then copies the cloned DNA using its own replication mechanisms. A variety of cell types are used a hosts, including bacteria, yeast cells and mammalian cells.
Polymerase chain reaction (PCR)
Another way of making many copies of a specific section of DNA, without the need for vectors or host cells, is through a polymerase chain reaction (PCR). The DNA to be copied - the template DNA - is mixed with two 20 base pair primers complementary to the end of the template DNA, nucleotides, and a version of DNA polymerase known as Taq polymerase. (This enzyme is stable under high temperatures, and is obtained from the thermophilic bacterium Thermus aquaticus.) The process involves the repetition of three steps:
- denaturation, which separates the two nucleotide strands of the DNA molecule
- primer annealing, in which the primers bind to the single-stranded DNA
- extension, in which nucleotides are added to the primers - in the 5' to 3' direction - to form a double-stranded copy of the target DNA
Each cycle takes about a few minutes, so repeated cycles can produce large amounts of a specific DNA sequence in hours rather than days. However, some details about the nucleotide sequence to be copied must be known in advance, and the technique is sensitive to small amounts of contamination.
A gene library is a large collection of cloned DNA sequences from a single genome. A genomic library, in theory, would contain at least one copy of every sequence in an organism's genome. To investigate the structure of a given chromosome, or to clone specific genes, libraries may be prepared from a subset of the entire genome (for example, a single chromosome). The first step is to break up, or 'fractionate', the genome using physical methods or restriction enzymes. The fragments are then linked to appropriate vectors and cloned in a suitable host cell population.
A cDNA library (complementary DNA) contains DNA prepared from the mRNA present in a given cell population using the enzymes reverse transcriptase, which produces single-stranded DNA from mRNA, and DNA polymerase, which converts single-stranded DNA into double-stranded DNA. The resulting cDNA represents the genes expressed in the cell population as a subset of the entire genome, and can be cloned using a vector and suitable host cell. The cDNA will not include introns or regulatory sequences as these are removed from the RNA during processing. A cDNA library can also be prepared using reverse transcriptase PCR (RT-PCR).
The identification and analysis of genes and gene products
Restriction enzymes (to cut the DNA) and gel electrophoresis (to separate the resulting fragments) can be used to produce a physical map of DNA segments in a process known as restriction mapping.
There are also a number of techniques that can be used to identify specific genes or gene products within a gene library: Southern blotting detects the presence of particular nucleotide sequences in a collection of DNA fragments using a DNA probe - a section of DNA labelled using radioactivity or chemical fluorescence; Northern blotting investigates gene transcription by identifying specific RNA sequences using labelled DNA probes; and Western blotting detects specific proteins using labelled antibodies.
However, the most powerful experimental technique for investigating genetics at the molecular level is DNA sequencing, which allows the nucleotide sequences of genes - even whole chromosomes - to be determined. Similar techniques are available for analysing the nucleotide sequences of RNA molecules and the amino acid sequences of proteins. Automated sequencing technologies are now allowing us to sequence the entire genomes of organisms from bacteria to human beings.
Molecular genetics and biotechnology
The new techniques of molecular genetics, combined with developments in associated biotechnologies, have led to advances in a number of different fields. We can now analyse the genomes of species that make an important contribution to agriculture, fuel production or drug development. We can move specific genes from one organism to another to create transgenic plants and animals, and use animal cloning techniques to produce animals that are genetically identical.
The technique of genetic fingerprinting has found many applications, including the identification of individuals and the relationships between individuals. Research into gene therapy examines the possibility of introducing cloned genes to compensate for defective, mutant genes. In other areas - for example, human cloning and stem cell research - there are ethical issues that must be addressed alongside the scientific developments.