This technology arose in response to the need for specific genetic segments in sufficient quantities for biochemical analysis.
The method involves trimming the desired segment of the surrounding DNA and copying it millions of times.
The success of recombinant DNA technology, whereby microbial cells can be engineered to produce foreign proteins.
It is based on the faithful reading of the corresponding genes by the bacterial cell machinery and has fed most of the recent advances in modern molecular biology.
During the last twenty years, studies of cloned DNA sequences have given us a detailed knowledge of gene structure and organization.
These have provided clues about the regulatory pathways by which the cell controls gene expression in the multiple cell types that make up the basic vertebrate body plan.
Genetic engineering, by which an organism can be modified to include new genes designed with the desired characteristics, is now a standard practice in basic research laboratories.
It has provided the means to produce large quantities of normal proteins and highly purified mutants for a detailed analysis of their function in the organism.
Recent advances in this technology have also changed the course of medical research.
New and exciting approaches are being developed to exploit the enormous potential of recombinant DNA research in the analysis of genetic disorders.
The new ability to manipulate human genetic material has opened radically new paths for diagnosis and treatment, and has far-reaching consequences for the future of medicine.
However, the basic principles of recombinant DNA, such as the structure of DNA itself, are surprisingly simple.
Molecular cloning provides a means to exploit the rapid growth of bacterial cells to produce large quantities of identical DNA fragments, which by themselves do not have the capacity to reproduce.
The DNA fragment to be amplified is first inserted into a cloning vector. The most popular vectors currently in use consist of small circular molecules of DNA (plasmids) or bacterial viruses (phages).
The vectors contain genetic information that allows the bacterial DNA replication machinery to copy them. After the insertion of the foreign DNA, the plasmid or phage vector is reintroduced into a bacterial cell.
The growing bacterial culture replicates the foreign DNA, along with the vector, in hundreds of copies per cell. This process produces multiple and identical clones of the original recombinant molecule.
It is easy to harvest vectors from the bacterial culture and release the foreign DNA fragments amplified with the same restriction enzyme used to insert the original DNA fragment into the vector.
The power of molecular cloning is remarkable: a liter of bacterial cells designed to amplify a single human DNA fragment of clones can produce approximately ten times the amount of a specific DNA segment that could be purified from the total cellular content of the entire human body .
For the analysis of long stretches of DNA, eukaryotic vectors that can grow in yeast that can contain megabases of foreign DNA have been developed.
These vectors mimic the chromosomal structure of the yeast, so they replicate together with the native yeast chromosomes each time a yeast cell is divided.
Yeast Artificial Chromosomes, or YACs, are often the only way to clone extremely large genes, including huge introns, all in one continuous piece.
YACs also provide a way to propagate DNA in a eukaryotic cell , where the modification of DNA, an important part of the eukaryotic genetic regulatory machinery, is more likely to be retained (more on this later).
YACs are increasingly useful in many ongoing genomic projects, since we intend to understand the meta-structure of chromosomes, where the location and disposition of genes within the “junk” DNA that surrounds them may contain regulatory information not yet discovered for the packaging and accessibility.
Amplification of recombinant DNA
The DNA segment to be amplified is separated from the surrounding genomic DNA by cleavage of restriction enzymes, which often produces staggered or sticky ends.
In the example illustrated here, the restriction enzyme EcoRI recognizes the palindromic sequence GAATTC, and cuts in each chain between G and A (the two strands of the genomic DNA are green and purple).
The plasmid vector (brown) is prepared to accept the isolated genomic DNA fragment by cutting the circular plasmid DNA at a single site with the same restriction enzyme.
Generating adhesive ends that are complementary to the adhesive ends of the genomic DNA fragment.
The cut genomic DNA and the linearized plasmid are mixed together in the presence of an enzyme ligase, which binds to the bonds in the DNA backbone on each side of the junction of the plasmid-genomic DNA.
This recombinant DNA molecule is then introduced into the bacteria that can absorb the plasmid DNA and then replicate the plasmid as the culture grows.
Why is rDNA important?
Recombinant DNA has been gaining importance in recent years, and will only be more important in the 21st century as genetics diseases become more frequent and the agricultural area is reduced. Below are some of the areas where recombinant DNA will have an impact:
- Better harvests (drought and heat resistance).
- Recombinant vaccines (ie, hepatitis B).
- Prevention and cure of sickle cell anemia.
- Prevention and cure of cystic fibrosis.
- Production of coagulation factors.
- Insulin production
- Production of recombinant pharmaceutical products.
- Plants that produce their own insecticides.
- Germinal line and somatic gene therapy.
How does rDNA work?
Recombinant DNA works when the host cell expresses the protein of the recombinant genes.
The host will not produce a significant amount of recombinant protein unless it expresses factors are added.
The expression of protein depends on the gene being surrounded by a collection of signals that provide instructions for the transcription and translation of the gene by the cell .
These signals include the promoter, the ribosomal binding and the terminator. The expression vectors, in which the foreign DNA is inserted, contain these signals.
The signals are specific to the species. In the case of E. Coli, these signals must be E. Coli signals since it is unlikely that E. coli will understand the signals from human promoters and terminators.