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TOOLS FOR GENETIC ENGINEERING

1.Restriction Enzymes: For insolation of a DNA fragment and production of recombinant DNA, the DNAs are desired to be cut at specific sites by the restriction enzymes. These enzymes are named by 3 or 4 letter abbreviations that identify their origin, and the Roman numerals, I, II, III, etc., indicating the isolation of several types of enzymes from the same microorganism. The restriction enzymes obtained from various microorganisms recognize and cut the DNAs at specific sites that are short (4-8 base pairs long palindromic sequences) double-stranded nucleotide sequences. Different restriction enzymes recognize different specific sequences. For some restriction enzymes the recognition and cutting sites on the DNA are located at different positions, whereas, for others the recognition and cutting sites are the same (used in recombinant DNA technology) (Table 12.1). Among the restriction enzymes that are used in genetic engineering, some cut the double stranded DNA molecule at different locations (staggered cuts) to produce short complementary sticky ends that may reanneal (e.g. EcoRI obtained from Escherichia coli) (Fig. 12.3),

Fig-12.3

Fig 12.3 Restriction enzyme action of EcoRI

whereas, others cut the two strands of DNA at the same locations to produce blunt ends (e.g. HaeIII obtained from Haemophilus) (Fig. 12.4)

Fig-12.4

Fig.12.4 Restriction site of SmaI and restriction of enzyme action of HaeIII.

Table 12.1: Recognition sites and cut ends produced by some restriction enzymes.


a.  
Plastids: Plastids are the extrachromosonal, self-replicating, double-stranded circular DNA molecules (1-1000 in number) found in many prokaryotic cells and a few eukaryotic cells (e.g. yeast). They confer properties like, antibiotic cells and a few toxin production, etc., and are not essential for the survival of the host cells, e.g. pBR322 plasmid (Fig 12.5). All the suitable properties to be used as vector may not be present in a plasmid and they are modified by inserting the DNA segments imparting desired features (e.g. antibiotic resistance) or removing undesired parts. They can be easily isolated and transferred to other bacteria and can accommodate 10-15 kbp (kilo base pair) of DNA. The recombinant plasmids are transformed into a suitable host cell (e.g. E.coli) for multiplication. 2. Vectors: Vectors are the carrier DNA molecus (or microorganism) for introduction of foreign DNA into the host cell. These vectors multiply in their usual manner even after carrying the foreign DNA, so that the inserted foreign DNA also replicates along with the parent vector DNA. The vectors should possess a site (e.g. restriction site) for foreign DNA insertion without disrupting any essential function, a selection marker (e.g. antibiotic resistance) for identification of the recombinant DNA containing the foreign DNA, etc. Some important vectors used in recombinant DNA technology are:

 

Cosmids are the plasmids containing cos site of λ-phage that regulates the packing of DNA into phage particles and can accommodate 35-45 kbp of foreign DNA

Fig-12.5

b)     Viruses: Most commonly, bacteriophages (e.g. λ-phage that can accommodate 22 kbp of foreign DNA) are used as vectors that contain linear DNA molecules. The non-essential DNA parts of the phage DNA are removed so that the foreign DNA can be inserted into it to produce the recombinant DNA (the protein coat of the phage can accommodate only a definite length of DNA). The recombinant DNA is allowed to infect the host cell where it replicates to produce a large number of progeny phage particles each containing a foreign DNA fragment. In addition to the bacteriophages, certain plant (e.g. CaMV or Cauliflower Mosaic Virus, TMV or Tobacco Mosaic Virus –a RNA virus) and animal (e.g. SV-40 or Simian Virus-40) viruses are also used as vectors in plants and animals, respectively. The use of viruses is restricted as they have narrow host range

c)   Artificial Chromosomes: Certain chromosomes have been prepared artificially to be used as vectors that can accommodate several hundred kbp of foreign DNA (sometimes the whole chromosome), e.g. YAC (Yeast Artificial Chromosome) by isolating and cloning telomeres (terminal parts of chromosomes) and centromeres of yeast chromosome in a plasmid (Fig12.6), BAC (Bacterial Artificil Chromosome) etc.

Fig-12.6

b)     Agrobacterium: Agrobacterium tumefaciens bacterium, that cause crown gall disease on the stem of dicot plants, contains a plasmid called Ti plasmid (tumour inducing). This Ti plasmid has a T-DNA region into which the foreign DNA can be inserted. This T-DNA is transferred to the host plant cell and gets incorporated into the chromosome of host cells (Fig. 12.7 in Appendix and 12.8). Another species, A. rhizogenes causes hairy root disease in dicots and contains a plasmid, Ri plasmid (root-inducing plasmid). Ti and Ri plasmids are used as vectors in dicot plants. For cloning purpose, the disease-causing part of the plasmid is removed. Thus, after infection by the Agrobacterium carrying foreign DNA to its host plant cell (the cultured cells or the leaf discs, root sections, floral parts, etc.), though the foreign DNA is expressed, but, it does not cause any disease (crown gall/hairy root disease). The plant cells containing the foreign DNA are regenerated into complete plantlets. This technique is successful only in dicot plants and not in monocot plants as Agrobacterium does not infect monocot plants. In monocots, other technique e.g. direct gene transfer) are used for the transfer of foreign DNA.

Fig-12.8

Fig. 12.8 Ti plasmid of A. tumefaciens.

1.      Direct DNA Transfer:Several technologies for direct DNA transfer are used in plants and microbes:

a.      Gene Gun: Gold or tungsten particles coated with foreign DNA are projected into the target cells with the help or gene gun machine. After penetrating the cell wall or cell membrane, the DNA gets incorporated into the genomic DNA of the cell (Fig. 12.9). 

Fig-12.9

b)     Polyethylene Glycol (PEG): Polyethylene glycol (15-25%) stimulates the uptake of DNA by the protoplasts (cells without cell walls).

c)      Electroporation: Exposure of protoplasts to electric current for very short duration of time induces temporary pores in the cell membrane through which the DNA enters readily and gets incorporated into the genomic DNA of the cell.

d)     Microinjection: This technology involves the injection of DNA using micropipettes 0.5-
10µm tip diameter) into the target cell that is immobilized on a solid support (coverslip or slide).

e)      Macroinjection: Here, the DNA is injected using needles having diameter greater than the cell diameter. In rye (Secale cereale) DNA was successfully injected into stern below the floral meristem (actively dividing tissue) from where it reached the sporogenous cells to develop into the transgenic plants containing the foreign DNA.

f)       Liposomes: In this technique the DNA is encapsulated into lipid bags and then they are fused with the protoplasts so that the DNA enters into the cel1.

Some Important Techniques Used in Recombinant DNA Technology

1.      Gel Electrophoresis: In this technique, the DNA, after extraction, is cleaved into fragments of different length by treating with a restriction enzyme. An agarose (or polyacrylamide) gel slab ( about 10 cm long and 0.5 cm thick) is prepared by pouring hot solution of agarose and a buffer into a mould (tray). When still in molten condition, a comb is placed on one side of the slab, which is removed after solidification to produce wells in the agarose gel slab. This slab is then immersed in a buffer solution in a tray and the mixture of the DNA fragments are loaded into the wells with the help of a micropipette. Under electric field the DNA fragments, being negatively charged, start moving towards the anode. The speed of movement depends upon the molecular weight of the DNA fragments, so that the lighter fragments move faster than the heavier fragment. Thus, different fragments of ifferent lengths appear as various bands (Fig. 12.10).

Fig-12.10

Fig. 12.10 Gel electrophoresis apparatus (left) and agarose get with DNA bands (right).

2. Blotting: A mixture of DNA/RNA/proteins is separated by gelelectrophoresis (as bands)
and hybridized with a labeled probe. Then those bonds are transferred to a nitrocellulose membrane by putting it on the surface of gel and this technique is known as blotting. The blotting of DNA (hybridized with labeled DNA or RNA probe with radio-active 32P) bands is known as Southern blotting after the name of E.M.Southern, (Fig 12.11), whereas the blotting of RNA (hybridized with labeled DNA or RNA probe with radioactive 32P) is called northern blotting and of protein bands (on polyacrylamide gel bound with labeled antibody probe with radioactive 125I) is called western blotting. 

Fig-12.11

Fig. 12.11. Technique of Southern blotting.