Introduction of recombinant DNA into a suitable host
A. Increase competence of host for transformation
• Competent host is essential for transformation with the recombinant DNA. Transformation is a process by which a cell takes up the naked DNA fragment from its environment, incorporates it into its own chromosomal DNA, and finally expresses the trait controlled by the incoming DNA.
• Finally, the propagation of these DNA molecules must occur inside a living system or a host.
• Many kinds of host cells, including E.coli, yeast, animal and plant cells, are available for genetic engineering and the kind of host cell to be used mainly depends on the aim of the cloning experiment.
• For the expression of some eukaryotic proteins, eukaryotic cells may be the preferred hosts.
• Yeasts have been used extensively for functional expression of eukaryotic genes because they offer several advantages.
• Since DNA is a hydrophilic molecule, it can not pass through membranes, so the bacterial cells must be made capable to take up DNA.
• This is done by treating them with a specific ; concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cells wall.
• Recombinant DNA (rDNA) can then be forced into such j cells by incubating the cells with recombinant DNA on j ice, followed by placing them briefly at 42°C (heat ; shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.
• In eukaryotic cell, the term transformation is replaced by the term transfection.
Nucleic acid blotting techniques
• Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA fragments from thousands of molecules.
• Southern blotting technique is the first nucleic acid blotting procedure developed in 1975 by Southern.
• This technique is extremely specific and sensitive, although it is a simple technique.
• It is an invaluable method in gene analysis, important for the confirmation of DNA cloning results, forensically applied to detect minute quantities of DNA (to identify parenthood, thieves, rapists etc.), highly useful for the determination of restriction fragment length polymorphism (RFLP) associated with pathological conditions.
• Northern blotting is the technique for the specific identification of RNA molecules.
• It is theoretically, a good technique for determining the number of genes (through mRNA) present on a given DNA.
• Dot blotting is a modification of Southern and Northern blotting technique.
• This technique is particularly useful in obtaining quantitative data for the evaluation of gene expression.
• Western blotting involves the identification of proteins. It is very useful to understand the nucleic acid functions, particularly during the course of gene manipulations.
B. Vectorless gene transfer
• Different alternative methods have been used to : introduce the recombinant DNA into recipient cells of ; animals, without involving a cloning vector.
• In this method, foreign DNA is directly injected into the nucleus of animal or plant cell by using micro needles or micro pipettes. It is used in oocytes, eggs and embryos.
• In method, electrical impulses induce formation of transient (temporary) pores in the plasma membrane of host cells by using calcium chloride. These pores of the membrane are the areas through which the DNA molecules are incorporated into the host cells.
Gene gun or Biolistic Method
• DNA is coated onto microscopic gold or tungsten pellets of size 1-2 mm, which are literally, shot with high velocity into the target cells as microprojectiles.
• Although this technique is developed for plants, it is also used to insert genes into animal cells, that promote tissue repair.
Direct DNA injection
• Direct DNA injection into skeletal muscle led to the possibility of using genes as vaccines. Due to low level of expression, therapeutic benefits for the treatment of genetic disorder could not be derived. This method gave birth to the concept of DNA vaccine or genetic immunization.