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1. DNA-DNA hybridization/rDNA technology:
In general when a double stranded DNA is heated, the two strands separate into single stranded molecules.
On cooling re-association of the two complementary strands slowly takes place, these results in the formation of the original double stranded DNA complex.
If single stranded DNAs from two different microorganisms are mixed and the nucleotide sequences are identical or nearly identical DNA-DNA hybridization takes place.
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The DNA-DNA hybridization method has been used for the following purposes:
(a) Detecting relationship of two microorganisms.
(b) Detecting similarities in the base sequence between nucleic acids of two different strains.
The schematic as shown in Fig. 2.10 shows that in:
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(a) The two nucleic acids are related where as in
(b) Only a portion of the nucleic acids are homologous.
It appeared that the hybridization procedure becomes easier and works more efficiently if one of the hybridizing nucleic acids is in fairly small pieces; hence one of the nucleic acids is subjected to high speed shearing treatment in a blender.
2. Cloning:
According to Mendel’s rules of inheritance of biological characteristics each inheritable property of an organism is controlled by a factor, called a gene which resides on chromosomes. In practice of molecular cell biology (MCB), cloning refers to gene cloning.
The definition of cloning involves following five steps:
1. Selecting and fragmenting proper DNA: The fragmenting of DNA containing the gene to be cloned is inserted into a circular DNA molecule called a vector to produce a chimera or recombinant DNA molecule (r DNA).
2. The constructed vector (vehicle) acts to transport the gene into a host cell to be redesigned which is usually a bacterium, although other type of bio cells can be used.
3. The vector multiplies inside the host cell producing numerous identical copies not only of itself but also of the gene that it carries.
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4. During host cell division copies of the rDNA molecules are passed to the progeny and further vector replication takes place.
5. On completion of a large number of cell divisions, a colony or clone, of individual identical host cells is produced. Each cell in the clone contains molecule, the gene carried by the redesigned cell is now said to be cloned.
3. Conjugation/Cell fusion:
This method is one way of bringing together unaccustomed cell partners, i.e. protein coding genes and control regions.
This method of redesigning cell system aims at the following:
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(a) Attachment of one of the in-expressed genes to a control region.
(b) Turning it on under the conditions prevailing inside the bioreactor/fermentation vessel.
(c) Making provisions for the silent genes became expressed-mRNA being copied from the gene and enzymes being synthesized which the microbe may use to manufacture new and useful product or over production of chemicals like antibiotics and amino acids.
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A typical cell fusion strategy is as below:
Cells
Remove the outer membrane of the cells by suitable lytic enzyme
Protoplast
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Fuse with protoplast of other cell
Hybrid or Recombinant cell
Contains genetic matter of two (or more) cells which is different from the previous individual thus redesigned.
4. Transfectional molecular blending:
Biochemical engineers have been successful to reconstruct bio toxins by molecular blending for unusual therapeutic application of these molecules in hospitals. Many cells are known to express the desired protein for a period of several days after transfectional molecular blending. In prokaryotic bio cells this is spoken of if bacteria form infectious phage progeny after the uptake of free phage DNA.
Two prerequisites have been stated to be required for this:
(1) The cells must be able to take up nucleonic acids of high molecular weight (the cells must be competent),
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(2) One entire phage entire the cell. Sometimes redesigning/reconstruction of an entire genome occur by recombination of overlapping fragments. Transfectional bio cell virus interactive process biotechnological production method of viral vaccines, hormones and interferon’s are good examples of this method.
5. Stress induced in vivo molecular signaling:
In vivo redesigning possibilities of newer bio products by stressing of bio cells are now known. In vivo redesigning of biomolecules using interactive stresses has been shown to produce stress proteins of commercial importance. Much more research is under way in producing, designing and promoting stress proteins of commercial beneficial use. Powerful modern instruments and techniques such as NMR, ESR, HPLC, PAGE, 2D-gel chromatography, GCMS etc. in conjunction with modern techniques are being used.
