BASIC PRINCIPLES OF GENETIC ENGINEERING
Genetic engineering involves manipulation of the genetic material towards a desired end in a direct and pre-determined way. This is alternatively called recombinant DNA technology or gene cloning. It started to develop in the mid 1970s.

Basic steps involved:
In short, gene cloning is essentially the insertion of a specific piece of 'desired DNA' into a host cell in such a way that the inserted DNA is replicated and handed onto daughter cells during cell division. The main factors involved in gene cloning are the following
• Isolation of the gene to be cloned.
• Insertion of the gene into another piece of DNA called vector which will allow it to be taken by bacteria and replicated within them as the cells grow and divide.
• Transfer of the recombinant vector into bacterial cells, either by transformation or by infection using viruses.
• Selection of those cells which contain the desired recombinant vectors.
• Growth of the bacteria, that can be continued indefinitely, to give as much cloned DNA as needed.
• Expression of the gene to obtain the desired product.


ISOLATION OF DNA FRAGMENTS:
The desired DNA fragments can be isolated by means of four mechanisms.
• Restriction endonuclease digestion:
It uses the restriction enzymes to cleave the desired region of the DNA. They are a group of enzymes that recognizes specific nucleotide sequences in DNA, often 4 or 6 base pairs long, and cut both strands of DNA within the recognition site. They are site specific.

Two types of cuts are made by these enzymes:
Blunt ends
If it cleaves both DNA strands at precisely opposite points on the two strands, it leads to blunt end fragments which are difficult to ligate or join to the vector in the next step.
Cohesive ends
In some cases the two DNA strands are not cut directly at opposite points, instead, the cuts are staggered forming cohesive ends (sticky ends). Sticky ends are best suited for cloning purposes as the staggering ends facilitate binding of another piece of DNA.

• Mechanical shearing :
It is done by sonication (use of sound waves to shear the DNA) or by forcing the DNA molecule using a syringe.

• Duplex cDNA synthesis:
Some times it is possible to synthesize a complimentary DNA (cDNA) strand to that of the desired DNA. It is done by two methods:

Classical method - here oligonucleotide dT primers, klenow fragment of T4 DNA polymerase and S1nuclease is used to synthesize cDNA.

New method - here terminal transferase and dCTP primer is used. After removing any contaminating mRNA by sucrose gradient, oligo dGTP primeris added to synthesize the second DNA strand.

• Direct chemical synthesis:
The desired DNA fragment can be synthesized if the sequence of the desired DNA is known.


INSERTION OF THE DESIRED GENE INTO A VECTOR

Once the desired DNA fragment is obtained it has to b transferred to the host cell. Cloning vehicles are small plasmids, phage or (animal virus DNA molecules) used to transfer a DNA fragment into a living cell. Cloning vehicles are also called vectors. They should have the following properties:
• Origin of replication to enable independent replication.
• Presence of recognition sites for restriction enzymes for insertion of the DNA fragment.
• Must be able to replicate in host cell after transfer.
• Presence of several markers for selection / screening.


The desired gene can be inserted or ligated into the vector by different methods:

Homopolymer tailing:
Here same bases are added to the terminal end, for example 8 mol of poly G tail is added by means of terminal transferase. Thus the complementary strand synthesizes a poly C tail. So the incoming DNA need not be cut.


Linker molecule:
In case of absence of restriction enzymes site, a short sequence which bears a site for a
specific restriction enzyme is introduced and ligated to the DNA by the enzyme DNA ligase. For non-complementary single strand, linkers are used along with adaptors.
Adaptors are self complement try.

Blunt end ligation:
In case of blunt ends in the DNA and the vector, high concentration of both plasmid and insert DNA is required and DNA ligase is used to ligate them. Self ligation is found to occur in low concentration.

Ligation of cohesive terminals:
This is more effective and naturally occurring


INTRODUCTION INTO THE HOST CELL
Once the vector and the desired DNA molecule is ligated, it has to be transferred to a host cell where it would replicate and produce copies of the desired gene and consequently its products. This transfer can be achieved by the following methods.
Transfection with recombinant phage DNA:
In case the vector being a phage it can infect the host cell and thus transfer the gene into the host.

Transformation with recombinant plasmid:
In case of the vector being a plasmid it can be transferred to the host by recombination.

SELECTION OR SCREENING
After the recombinant DNA is transferred to host via the vector, it is integrated into the host cell DNA and starts replicating along with the host or is replicated independently along with the phage within the host. In both the cases the host cells become factories where the desired gene is replicated and expressed. The step thereafter includes screening of the host cells to check for successful integration and replication of the desired gene and expression of its products. This is achieved by the following methods:


GENETIC METHOD:
This involves the expression of certain traits. Usually these traits are encoded by the vector or perhaps by the desired cloned sequence if a direct selection method is available. One of the simplest methods involves the use of antibiotics to select for the presence of vector molecules. Eg: pBR322 carries genes Ampr and Tcr which confer resistance to Ampicillin and Tetracycline respectively.

SCREENING USING NUCLEIC ACID HYBRIDIZATION:
It is a very powerful method of screening clone banks, and is one of the key techniques in gene manipulation. It uses a defined nucleic acid probe which will identify the presence of the desired gene sequence. The power of nucleic acid hybridization lies in the fact that complementary sequence will bind to each other with a very high degree of fidelity. Three main types of probes are used - cDNA , genomic DNA, oligonucleotides.

IMMUNOLOGICAL SCREENING:
Here the protein product of a cloned gene is identified by immunological method. Instead of a nucleic acid probe, a specific antibody is used. Detection may be by radioactive or non radioactive method.

ANALYSIS OF CLONED GENES:
This method involves the identification of the protein product by two methods based on translation of mRNA in vitro. These methods are known as Hybrid release translation (HRT) and Hybrid arrest translation (HART). HRT is the preferred method.

BLOTTING TECHNIQUES
Here the samples are first made to run in a gel electrophoresis,
the separated fragments are then transferred to a nitrocellulose or nylon membrane by a blotting technique. The original method uses capillary technique. The filter can then be hybridized with a radioactive probe. After hybridization the filter is washed and later, exposed to X- ray film and an autoradiogram prepared, which provides information on the structure of the clone.

SOUTHERN BLOTTING:
It is used for running DNA samples. This was first developed by Ed southern hence the name. Here agarose gel is used.

NORTHERN BLOTTING:
Here RNA samples are been made to run. Here also agarose gel is used.
WESTERN BLOTTING:
It used to find the proteins. Here SDS PAGE method is followed. Membrane is then probed with an antibody to detect the protein.

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