The following points highlight the four main techniques used for gene cloning. The techniques are: 1. Isolation of DNA to be Cloned 2. Insertion of Foreign DNA Fragment into a Vector 3. Transfer of Recombinant DNA into Bacterial Cell 4. Detection of Recombinant Clone.
Technique # 1. Isolation of DNA to be Cloned:
The DNA of interest, i.e., target DNA may be genomic DNA or complementary DNA or synthetic DNA. The genomic DNA of interest if contained in a particular restriction fragment, that can be isolated from gel after electrophoresis.
Otherwise, a complementary DNA (cDNA) fragment is prepared directly by using mRNA as template. The polyadenylated mRNAs are separated from other types of RNAs through affinity column chromatography.
These mRNAs are then copied to cDNAs with the help of reverse transcriptase. In these cases as the cDNA is obtained from mRNA, so it must contain the uninterrupted coding sequence of gene and the recombinant DNA molecule will synthesize the eukaryotic gene product in prokaryotic cell. One can also synthesize the desired DNA fragment by machine.
Technique # 2. Insertion of Foreign DNA Fragment into a Vector:
The cDNA thus isolated above or obtained from gene bank is fragmented by using the specific restriction enzyme to develop specific cohesive ends. The cloning vector is also treated with the same restriction enzyme, so that the cohesive ends are generated (Fig. 18.5).
For insertion of double stranded cDNA into a cloning vector, it is necessary to add to both termini single stranded DNA sequence which should be complementary to a tract of DNA at the termini of linearized vector. In order to get efficient formation of recombinant DNA molecules, addition of sticky ends on both termini is necessary.
There are two methods for generation of cohesive ends on the double stranded cDNA:
(i) Use of linkers
(ii) Homopolymer tails.
Linkers are the chemically synthesized double stranded DNA oligonucleotides containing on it one or more restriction sites for cleavage by restriction enzymes (Fig. 18.6).
Linkers are ligated to blunt end by T4-DNA ligase. Using terminal transferase the synthesis of homopolymer tails of the defined length at both 3′ termini of double stranded DNA and vector is possible. If the poly- T tail is added at the termini of foreign DNA, then poly-A tail is added at the restriction site of the vector, so that the complementary sticky ends are formed and they get annealed by T4-DNA ligase (Fig. 18.7).
Technique # 3. Transfer of Recombinant DNA into Bacterial Cell:
Before the recombinant DNA can be bulked up by cloning, it must be taken up by a suitable bacterial host cell, which is then said to be transformed, i.e., a host bacterial cell must accept the plasmid with the foreign gene, get it incorporated into its genome and start transcribing that gene.
The event of entering the plasmid with foreign DNA into the cell is known as “transformation”. A mild heat shock is given to the mixture which results in the uptake at higher frequency of the DNA. The selection of transformed cells is done by allowing the bacteria to grow in antibiotic selection medium.
Cloning in Eukaryotes:
In eukaryotes the nucleus is separated from the rest of cell through nuclear membrane, many of the genes are split genes with exons and introns. As such genetic engineering with eukaryotes needs special methods.
When eukaryotic genes are cloned in prokaryotes, the split genes cannot be correctly expressed, because prokaryotes do not have the machinery for splicing out the RNA transcribed from the introns of a gene. So the eukaryotic cells are needed for cloning and expression of cloned eukaryotic genes.
Among eukaryotes, DNA cloning has been done in yeast, mouse and in higher plant species. In yeast, a 2µ plasmid DNA is an appropriate cloning vehicle, which can be transferred through efficient transformation method. This involves protoplast production followed by PEG directed introduction of DNA into protoplasts.
Technique # 4. Detection of Recombinant Clone:
From the large number of colonies produced by transformation to select or screen out the few colonies which contain the recombinant plasmid — the use of antibiotics is one of the most easy and useful methods for this purpose. The transformed cells can be plated on selection medium containing different antibiotics.
The colonies which grow, can be said to have a plasmid, as the antibiotic resistance gene of plasmid enables the bacteria to grow. For example, the plasmid pBR 322 contains genes for ampicillin resistance (ampr) and tetracycline resistance (tetr). Thus the trans-formants can be detected by their plating potential on medium containing either (or both) of these antibiotics.
The presence of cloned DNA fragments can be detected by insertional inactivation of suitable genetic system. For example, DNA fragment of interest can be inserted into one of the antibiotic- resistance genes (tetr) of pBR322, inactivating that gene (tets) and other remains active (ampr).
To selectively kill cells with antibiotics, the original master plate with ampicillin in medium is subjected to replica plating method with both ampicillin and tetracycline. Bacteria with recombinant plasmid do not grow on replica plate, only with non-recombinant plasmid will grow. Recombinant colonies are thus identified and selected from master plate (Fig. 18.8).
The detection of recombinant clones can also be done by using chromogenic substrates. The most popular system uses X-gal, a colourless substrate on cleavage by β-galactosidase, a blue coloured product is formed, then the expression of lac Z gene can be detected easily.
Host cells that are Lac– are used, so that the Lac+ phenotype will only arise when the vector is present. Furthermore, if a DNA fragment is cloned into lac Z gene (Eco RI site of Charon 16A), any recombinants will be lac Z and therefore will not produce β-galactosidase and plaques will remain colourless in presence of X-gal.