RECOMBINANT DNA TECHNOLOGY
Using Recombinant DNA technology, we can isolate and clone single copy of a gene or a DNA segment into an indefinite number of copies, all identical. These new combinations of genetic materials or Recombinant DNA '(rDNA)' molecules are introduced into host cells, where they propagate and multiply. The technique or methodology is called Recombinant DNA technology or "Genetic engineering".
The first recombinant DNA molecules were generated by Paul Berg, Herbert Boyer, Annie Chang, and Stanley Cohen in 1973.
To obtain rDNA steps involved are:
a) The DNA fragment containing the gene sequence to be cloned (also known as ('insert') is isolated.
b) Insertion of these DNA fragments into a host cell using a "vector" (carrier DNA molecule).
c) The rDNA molecules are generated when the vector self replicates in the host cell.
d) Transfer of the rDNA molecules into an appropriate host cell.
e) Selection of the host cells carrying the rDNA molecule using a marker.
f) Replication of the cells carrying rDNA molecules to get a genetically identical cells population or clone.
Enzymes used in Recombinant DNA technology
For cloning of DNA, the DNA is cut at specific sites, which are recognized and cleaved by specific enzymes. These enzymes are known as restriction enzymes. These restriction enzymes recognize short sequences of double stranded DNA as targets for cleavage. Different enzymes recognize different but specific sequences, each ranging from 4-8 base pairs. The enzymes are named by a three letter (or four letter) abbreviation that identifies their origin e.g. AluI is derived from Arthrobacter luteus, EcoRI is derived from E.Coli, HpaI is derived from Haemophilus parainfluenzae
Besides cleavage, modification in the form of methylation is also brought about by some enzymes called modification enzymes sometimes also called “methylases”. Different species of bacteria contain their own sets of restriction enzymes and corresponding methylases. Depending on the different modes of action, the restriction enzymes have been classified into three main classes- type I, type II, type III. Out of these, type II restriction enzymes are used in recombinant technology as they can be used in vitro to recognize and cleave with in specific DNA sequences usually consisting of 4-8 nucleotides.
These enzymes form phosphodiester bonds between the adjacent molecules and, covalently links two individual fragments of double stranded DNA. The most commonly used enzyme for cloning experiment is T4 DNA ligase.
The enzyme alkaline phosphatase (AP) removes the phosphate group from the 5’ end of a DNA molecule leaving a free 5’ hydroxyl group hence it is used to prevent unwanted self-ligation of vector DNA molecules in cloning procedures. This enzyme is isolated from bacteria (BAP) or calf intestine (CAP).
Cloning vectors for Recombinant DNA
Vectors are the vehicles used to carry a foreign DNA sequence into a given host cell. A vector should have a) origin of replication, b) a selectable marker to select the host cells containing the vector from among those which do not have the vector, c) one unique restriction endonuclease recognition site to enable foreign DNA to be inserted into the vector in order to generate a recombinant DNA molecule and, d) it should be relatively small in size.
Plasmids as vectors
Plasmids are defined as autonomous elements, whose genomes exist in the cell as extrachromosomal units. They are self replicating, circular duplex DNA molecules present in number of copies in a bacterial cell, yeast cell or in organelles in eukaryotic cells. These naturally occurring plasmids have been modified to serve as vectors in the laboratory.
One of the most commonly used cloning vector in gene cloning procedures is pBR322 (named after Bolivar and Rodriguez who constructed this) derived from E. coli plasmid ColE1. It is 4,362 bp DNA with genes for resistance against two antibiotics- tetracycline and ampicillin. It was constructed after several alterations and modifications in earlier cloning vectors.
The plasmids belonging to pUC series of vectors are 2,700 bp long with ampicillin resistance gene, the origin of replication derived from pBR322 and the lac Z gene derived from E.coli. When the DNA fragments are cloned in this region of pUC, the lac gene is inactivated.
Yeast plasmid vectors
Special vectors used to introduce DNA segments in yeast cells or a eukaryotic cell is being used to develop genetically engineered yeast cells. E.g. YIp or yeast integrative plasmids which allows transformation by crossing over and have no origin of replication. YEp or yeast episomal plasmids carry 2 micron DNA sequence including the origin of replication and rep gene. These vectors have been widely used to study yeast genome.
The vectors containing two types of origin of replication which helps them to exist in both eukaryotic cell as well as E.coli are known as shuttle vectors. E.g.Yep vector.
A class of vectors which are used to retrieve specific genes from the normal chromosome of an organism like yeast through recombination. They are very useful in isolation of genes from yeast for molecular experiments like sequencing.
Vectors based on bacteriophages
Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. They are used as vectors because they have a linear DNA molecule, which generate two fragments after breakage. These are later joined with foreign DNA to generate chimeric phage particle. The injected DNA is selectively replicated and expressed in the host cell resulting in a number of phages which burst out of the cell (lytic pathway) and further infect the neighbouring cells. E.g. M13 , Lambda ( )
Cosmids as vectors
Cosmids are plasmid particles into which specific DNA sequences like cos sites of phage lambda are inserted and they are constructed by combining certain feature of plasmids and the ‘cos’ sites of phage lambda. The advantage of the using cosmids for cloning is that its efficiency is high to produce a complete genomic library of 10(6)-10(7) clones from only 1 microgrm of insert DNA.
Phagemids as vectors
Phagemids are prepared artificially by combining features of phages with plasmids. E.g. pBluescript II KS is derived from pUC19 and is 2961 bp long.
BACs or Bacterial Artificial Chromosomes are vectors based on the natural, extra-chromosomal plasmid of E.coli- the fertility or F-plasmid, and are being used in genome sequencing projects. A BAC vector contains genes for replication and maintenance of the F-factor, a selectable marker and cloning sites and can accommodate up to 300-350 kb of foreign DNA.
Plant and animal viruses as vectors
A number of plant and animal viruses can also be used as vectors for introducing foreign genes into cells and/or for gene amplification in host cells. Plant viruses like Gemini viruses, cauliflower mosaic virus or CaMV and tobacco mosaic virus /TMV) are three group of viruses that have been used as vectors for cloning of DNA segments. CaMV has a double stranded DNA molecule, 8kbp in size which infects the members of Cruciferae family. Geminiviruses are a group of single stranded DNA plant viruses infecting cassava, maize and other cereals.
Animal virus vectors also deliver the foreign genes into the cultured cells which get integrated into the host genome. The expression of foreign genes can also be amplified using the promoters of the virus gene. The cloned genes can be used in gene therapy, for the synthesis of important proteins etc. A vector based on Simian Virus 40 (SV 40) was used in the first cloning experiment involving mammalian cells in 1979. Retroviruses like murine and avian retroviruses are very useful vectors as they are capable of infecting a large variety of cell types and can clone large genes. Herpes virus is a non-integrating large sized virus (150 kb) which is another useful vector which can be used for expression of large intact genes.
Artificial chromosome (YAC and MAC) vectors
YACs or Yeast Artificial Chromosomes are used as vectors to clone DNA fragments of more than 1Mb in size. These long molecules of DNA can be cloned in yeast by ligating them to vector sequences that allow their propagation as linear artificial chromosomes. These long DNA molecules can be generated and allow construction of comprehensive libraries in microbial hosts. A lot of work is going on to create mammalian artificial chromosomes (MACs) following the isolation of mammalian telomeres and centromere. However YACs have a low cloning efficiency (1000 clones/microgm) DNA as against 106-107 clones/microgm DNA for cosmids) and also it is difficult to recover large amount of pure insert DNA from individual clones.
Polymerase Chain Reaction
A very small sample of DNA can be magnified many times using the technique of Polymerase chain reaction or PCR. This is the technology that helped to sequence the complete the human genome and also complete the Human Genome Project ahead of time. It was possible to develop this technique because of the discovery of a polymerase that was tolerant of high temperature called Taq. This polymerase was extracted from a bacterium Thermus aquaticus usually found in the hot springs of Yellowstone National Park. Using this polymerase now millions of copies of a single DNA segment can be produced in a very short period of time.
Before cell division, most organisms copy their DNA in each chromosome. For this the first step is to unzip the DNA chains of the double helix. When the two strands separate, the DNA polymerase starts copying each strand using them as template. DNA polymerase uses four nucleotide bases and a primer which is a short sequence of nucleotides to prime the process to start the DNA replication. The cell uses an enzyme called primase that helps to make the first few nucleotides of the copy. Once the primer is made the DNA polymerase makes the rest of the new DNA chain.
The process of Polymerase chain reaction does the same only in a test tube rather then a cell. Now a PCR vial is commercially available and the technique is used routinely in almost every molecular biology laboratory. A PCR vial has all the contents needed for DNA duplication- a piece of DNA; large quantities of the four nucleotides: A, T, G and C; a substantial quantity of the primer sequence, and Taq polymerase obtained from the bacteria Thermus aquaticus.
The process of PCR involves three steps. In the vial, the first step involves the heating of the target genetic material to 90-96C (165 F) for 30 secs to denature the material, which unwinds and unzips the two strands of the double helix DNA.
The second step involves the cooling of the vial to 55C. In this step, the short sequences of the primer are attached to the end of the single stranded DNA strands obtained by denaturing the DNA in the first step. As the primers cannot bind to the DNA strand at high temperature (90C), the vial is cooled to 55C for the second step where the primer binds and anneals to the ends of the DNA strands which takes about 20 secs.
In the third or the final step, polymerase reads a template strand and matches it with complementary nucleotides very quickly. The polymerase makes the two new DNA double strands form the original one. In the vial, as the Taq polymerase works best around 75C which is the temperature of the Hot springs where the bacteria is found, the temperature is raised to 75C. The Taq polymerase begins adding nucleotides to the primer and eventually makes a complementary copy of the template. If the template contains an A nucleotide, the enzyme adds on a T nucleotide to the primer. If the template contains a G, it adds a C to the new chain, and so on to the end of the DNA strand. This completes one PCR cycle.
All the three steps of the Polymerase Chain reaction or a PCR cycle involving the separation of the strands, synthesis and annealing of the primer to the template and the synthesis of the new strands completes in approximately 2 minutes. The cycle is repeated 30 or more times using the newly synthesized DNA using as a new template.
Using the technique, 1 billion copies of a single piece of DNA can be produced in a short period of time.