Recombinant DNA Technology
Recombinant DNA technology is the process of removing the desired piece of DNA from one organism and incorporating it into chromosomal DNA of another organism. It is also termed as genetic engineering.
Tools of Recombinant DNA Technology
The tools include enzymes termed as restriction endonucleases and also DNA polymerases and DNA ligase. The procedure also uses vectors as vehicles to carry foreign DNA into a host cell or living system. All these form tools for recombinant technology.
Restriction enzymes occur in bacteria where restriction enzyme or restriction endonuclease forms a major part of bacterial defence system by restricting the growth of potentially harmful genetic elements (i.e., DNA) of other bacteria, viruses (bacteriophages) etc.
Nuclease is an enzyme which breaks phosphodiester bonds between nucleotide subunits of nucleic acids. These are of two types exonucleases and endonucleases. Exonucleases work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds occurs at either the 3’ and 5’ end. Endonuclease on the other hand cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain at specific locations.
Molecular scissors/Molecular scalpels
Restriction endonuclease recognises a specific DNA sequence and degrades any DNA containing that sequence. These enzymes differ with different bacterial species. There are three types of restriction enzymes, Type I, Type II and Type III. Restriction enzymes Type II are useful in Recombinant DNA technology.
The credit for isolation of first endonuclease goes to D. Nathans and H. O. Smith (1970). They were the Nobel Prize winners in Physiology and Medicine for this discovery in 1978. These enzymes are now variously termed as ‘molecular scalpels’, ‘molecular scissors’, ‘chemical scalpels’ etc.
The DNA segments cut by restriction enzymes are pallindromic, i.e., the nucleotide sequence of these DNA pieces read the same both backwards and forward, for example, Madam.
The naming of these enzymes follows certain norms. These are named after the bacterium from which these are isolated. The first letter of the name comes from the genus of the bacterium (in italics), then comes the first two letters of the species (also in italics), next is the strain of the organism and the last is the roman numeral indicating the order of discovery. For example, the enzyme Eco RI was isolated from the bacterium Escherichia (E) coli (co), strains RY (R) and was the first endonuclease (I).
Blunt ends and Sticky ends
The cut produced by restriction endonuclease is important. The resultant DNA sequence may either have blunt ends or sticky ends.
Many restriction enzymes produce blunt ends or flush ends which leave both strands of DNA exactly at the same nucleotide position, in the centre of recognition site. It cuts both DNA strands producing blunt ends.
Sticky or cohesive ends occur when restriction enzymes do not cut DNA at the same nucleotide position but cut the recognition sequence unequally. This produces short single stranded overhangs at each end. These are termed as sticky ends. The stickiness helps enzyme ligase to make the DNA pieces to join.
Other Enzymes Used in Cloning
Besides restriction endonucleases, recombinant DNA technology requires other enzymes. The two enzymes are DNA ligase and alkaline phosphatase.
This enzyme is useful in joining the bits of DNA. DNA ligase forms phosphodiester bonds between adjacent nucleotides and joins two fragments of double stranded DNA. The enzyme forms phosphodiester bond between hydroxyl group at 3’ carbon of one nucleotide with phosphate group at 5’ carbon of another nucleotide.
The enzyme is useful in removing the phosphate group from 5’ end of a DNA molecule so that a free 5’ as hydroxyl group is available for ligation. It, therefore prevents unwanted self ligation of vector DNA molecules in cloning.
Another important tool for recombinant technology is the organism for cloning termed as vector. The vector carries a foreign DNA sequence for producing its multiple copies. Bacterial plasmid and bacteriophages are the most useful vectors. This is because of the following reasons:
- Both of these are independent of the control of chromosomal DNA.
- Bacteriophage genomes occur in very large number in bacterial cells and
- The copies of the plasmids per cell range from only a few to hundred or even more.
Following essential features should be present in a DNA molecule to act as a cloning vector:
Origin of Replication (Ori):
This is a DNA sequence which serves as a starting point for replication. When a DNA fragment associates with ori, foreign DNA into the vector would also replicate inside the host cell. Some vectors possess origin which favours formation of high copy numbers and hence preferred.
Vector should also include a selectable marker which is a gene permitting identification of transformants from non transformants. Common selectable markers in E. coli include genes encoding antibiotic resistance such as ampicillin resistance or enzymes such as beta galactosidase (product of z gene of lac operon). A colour reaction is helpful in the identification of these genes.
The process of selection of transformant or recombinant due to antibiotic inactivation is very complex and involves time consuming procedures. A simple and alternative method, therefore, is necessary. A technique termed as insertional inactivation was helpful in overcoming this difficulty.
Insertional inactivation is a technique used in recombinant DNA engineering where a plamid (such as pBR322) is useful to disable expression of a gene.
The inactivation of a gene is brought about by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it.
An example is the use of pBR322, which has the genes that respectively encode polypeptides that confer resistance to ampicillin and tetracycline antibiotics. Hence, when a genetic region is interrupted by integration of pBR322, the gene function is lost but new gene function is gained.
Vector should possess a unique recognition site that would allow particular enzyme to cut only once. The restriction enzyme recognizes this site. If is there are more than one recognition sites in the vector, several fragments would be produced.
Multiple cloning sites (MCS)
Generally the vectors used possess unique recognition sites for several restriction enzymes in a small region of DNA. This is termed as polylinker or multiple cloning sites (MCS). Such a cloning site offers choice of restriction enzyme.
Unique restriction endonuclease recognition site enables insertion of foreign DNA into the vector for production of recombinant DNA. The foreign DNA inserts and ligates at a specific restriction site generally in antibiotic resistance gene.
pBR322 has genes for resistance against two antibiotics (tetracycline and ampicillin), an origin of replication and a variety of restriction sites for cloning of restriction fragments obtained through cleavage with a specific enzyme.
Foreign DNA is inserted at a site located in one of the two genes for resistance against antibiotics, so that it will inactivate one of the two antibiotic resistance genes. The insert bearing plasmid can be selected by its ability to grow in a medium containing only one of the two antibiotics and its failure to grow in a medium containing both antibiotics.
The plasmids carrying no insert on the other hand, will be able to grow in media containing one or both the antibiotics. In this way the presence of resistance genes against ampicillin and tetracycline allow selection of Escherichia coli colonies transformed with plasmids carrying the desired foreigns cloned DNA fragment.
Competent Host (for Transformation and Recombinant DNA)
The use of tools finally results in the formation of recombinant DNA. Recombinant DNA thus formed needs to be multiplied inside living system or a host. Many types of host cells are available for this purpose. The choice of host, however, depends upon the purpose of cloning E. coli, yeast, animal and plant cells have been variously used. E. coli is the most commonly used organism in recombinant DNA technology. It has many advantages, such as:
- It is easy to manipulate and grow in vitro.
- E. coli can accept a wide range of vectors.
- It doubles the number of cells every 20 minutes and
- E. coli reproduces r-DNA molecule as it does for its own DNA.
Yeasts too are very useful in recombinant DNA technology because of the following reasons:
- These are simplest eukaryotic organisms.
- Yeasts are unicellular and hence easy to handle.