Gene cloning: Techniques
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.
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