NEET Biology Notes Biotechnology Principles and its Applications Cosmids
Cosmids
Cosmids are the vectors, which can accommodate DNA segments up to 45 kbp. These are actually plasmid particles and ‘cos’ sites of phage lambak.
So, we can say that the cosmids allow the packaging of DNA in phage in vitro, thus, permitting their purification. Like plasmids, these cosmids also perpetuate in bacteria.
Phagemids
These are the plasmids with a fragment of filamentous phage DNA. Segments of foreign DNA cloned in them àre multiplied as plasmid vectors, but when these plasmid vectors are infected with a filamentous phage (helper phage), the phagemid genome present already behaves like a phage. As a result, the phagemid generates multiple copies of one strand of it and the associated DNA inserted in it.
Bacterial Artificial Chromosome (BAC) Vectors
These are vectors based on natural, extra chromosomal plasmid of E. coli, the fertility or F-plasmid. A BAC vector contains genes for replication and maintenaince of the F-factor, a selectable marker and cloning sites. These vectors can accommodate up to 300-500 kb of foreign. DNA and are also being used in genome sequencing projects.
Yeast Artificial Chromosome (YAC) Vectors
These are used to clone DNA fragments of more than 1 Mb in size, therefore, they have been exploited extensively in mapping the large genomes, e.g. in the Human Genome Project. These vectors contain the telomeric sequence, the centromere and the autonomously replicating sequence from yeast chromosomes. They also contain restriction enzyme sites and genes which act as selectable markers in yeast.
Ti Plasmid
A Ti (Tumour inducing) plasmid is a circular plasmid that often, but not always, is a part of the genetic material to plants. The plasmid has 196 genes that code for 195 proteins. There is one structural RNA. The plasmid is 206-479 nucleotides bp long, the GC content is 56% and 81% of the material is coding genes. There are no pseudogenes.The modification of this plasmid is very important in the creation of transgenic plants.
Competent Host
It is essential for transformation wjtji recombinant DNA. Many kinds of host cells, including!?. coli, yeast, animal and plant cells are available for genetic engineering. In animals the term frgpsformation is replaced by the term transfection. nUtgre are other methods to introduce foreign DNA into host cells.
These are briefly described below:
- Microinjection In this method recombinant DNA is directly injected into the nucleus of animal cell by using micro needles or micro pipettes.
- Electroporation In this method the electrical impulses induce transient pores in the plasmalemma through which the DNA molecules enter the cells.
- Direct DNA injection Direct injection of DNA into skeletal muscle led to the possibility of using gene as vaccines.
- Gene gun or biolistics DNA coated onto microscopic pellets is literally shot into target cells. Although it is developed for plants yet this technique is also used to insert into animal that promote tissue repair into cells near wounds leading to reduction of healing time.
Processes of Recombinant DNA Technology
Recombinant DNA technology involves the following steps:
- Isolation of DNA
It has to be done in pure form for the reaction of restriction enzymes. Use of enzymes like lysozyme is done in this step.
- Fragmentation and Separation of DNA
It is carried out by incubating the purified DNA molecules with suitable restriction enzymes to produce DNA fragments. These fragments can be separated by a technique known as gel electrophoreis.
- Amplification
Amplification of the DNA/gene of interest refers to the process of making copies of the DNA segment in vitro. It involves Polymerase Chain Reaction (PCR). The PCR process was designed by K Mullis in 1983. He won Nobel Prize in 1993.
PCR involves three mains steps
- Denaturation
- Primer annealing
- Extension of primers.
- The double stranded DNA is denatured by using high temperature. Two sets of chemically synthesised primers are used in this process (PCR).
- Ligation
Ligation of amplified DNA fragment with the vector forms recombinant jDNA (rDNA). rDNA is made in first meiotic prophase dapfe process of crossing over.
- Transfer ofltaoiMMbmanl DNA
Transfer of reoewafeinant DNA . into host is done by microinjection method. Gene gun or biolistics as a suitable method for cells transformation.
- Culturing of Transgenic Cell
Cells containing the foreign gene are then cultured on a suitable medium to make clones.
- Extraction of Desired Product
Extraction of desired product is done by suitable procedure like by using bioreactors.
- Downstream Processing
- It is a series of processes, which are to be followed before the extracted product is ready for marketing.
Two main processes under this are - Separation
- Purification
- The product is then formulated with suitable preservatives and some products undergo clinical trials. This process is performed in large vessel called as bioreactors.
Bioreactor is a kind of vessel, in which raw materials are biologically converted into specific products by microbes, plant and animal cells and/or their enzymes. The most commonly used bioreactor is of stirring type.
A bioreactor has following components :
- An agitator system
- An oxygen delivery system
- A foam control system
- A temperature control system
- pH control system
- Sampling ports.
Random Amplification of Polymorphic DNA(RAPD)
It is a type of PCR reaction, but the segments of DNA that are amplified are random. The scientists performing RAPD with various short primers (8-12 nucleotides} randomly then proceeds with the PCR using a large template of genomic DNA with hope to amplify, fragments.
Restriction Fragment Length Polymorphism (RFLP)
In molecular biology, RFLP is used in two related contexts as a characteristic of .DNA molecules (arising from their different nucleoiidessequence) by which, they may be distinguislftddl aand as the laboratory technique which uses thiSqfibaracteristic to compare DNA molecules. This technique is utilised in genetic fingerprinting and paternity testing.