What is Genetic Engineering and steps involved in the process of genetic engineering?

GENETIC ENGINEERING

Genetic engineering is the process of altering the DNA in an organism’s genome. Scientists use genetic engineering to enhance or modify the characteristics of an individual organism. Genetic engineering can be applied to any organism, from a virus to a sheep.

STEPS OF GENETIC ENGINEERING

The job of a gene engineer is to identify and isolate the gene of interest and then have it introduced and joined in the genome of another organism, wherein it can either be cloned or allowed to express itself to produce the desired gene product. The steps involved in genetic engineering can be enumerated as:

1. Isolation of gene of interest, which is referred to as foreign or target gene.
2. Insertion of the isolated gene into a vector to form a recombinant DNA (rDNA) molecule.
3. Introduction of rDNA into a host cell.
4. Identification of and isolation of the transformed cells, i.e., cells that have taken up the rDNA (foreign gene + vector).
5. Cloning, i.e., replication of rDNA to produce multiple copies of itself.
6. Expression of the foreign genes to obtain the desired gene product.

1. ISOLATION OF THE GENE OF INTEREST:

The gene of interest that is to be isolated may be of bacterial, plant, or animal origin, or it can even be a newly (invented) synthesized gene. Four mechanisms can isolate the desired DNA fragments.

• Restriction endonuclease digestion: It uses restriction enzymes to cleave the desired region of the DNA. They are site-specific. These enzymes make two types of cuts 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 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 the binding of another piece of DNA.

• Mechanical shearing: The DNA is subjected to high-speed mixing and sonication (use of sound waves to shear the DNA) to generaterate target fragments of DNA. • Duplex cDNA synthesis: Sometimes, it is possible to synthesize a complementary DNA (cDNA) strand to the desired DNA. Two methods do it:
  • Classical method - oligonucleotide dT primers, Klenow fragment of T4 DNA polymerase, and S1 nuclease are used to synthesize cDNA.
  • The new method - here, terminal transferase and dCTP primer is used.

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

1. INSERTION INTO VECTOR:

Once the desired DNA fragment is obtained, it has to b transferred to the host cell. The desired gene can be inserted or ligated into the vector by different methods:

• Homopolymer tailing: UUsing a RE, if blunt ends are generated, the homopolymer tailing technique is useful for inserting the desired gene into a vector. Here same bases are added to the terminal end, and this technique involves using terminal deoxynucleotidyl transferase (TdT).
• Linkers and adapters: Another tech. By which blunt ends can be converted to have cohesive ends involves the use of linkers and adapters. An adapter, adaptor, or linker in genetic engineering is a short, chemically synthesized, single-stranded or double-stranded oligonucleotide that can be ligated to the ends of DNA or RNA molecules.
• RE generating blunt ends: Ligation of desired gene and vector having blunt ends can be brought about by using a high concentration of DNA ligase than required for ligating cohesive ends. It is the T4 DNA ligase that has to be employed rather than E. coli DNA ligase to join blunt ends.

1. INTRODUCTION OF rDNA INTO A HOST CELL:

• Once the vector and the desired DNA molecule areare ligated, it has to be transferred to a host cell to replicate and produce copies of the desired gene andits products. The following methods can achieve this transferfection with recombinant phage DNA: If the vector being used is a phage, it can infect the host cell and thus transfer the gene into the host.
• Transformation with recombinant plasmid: If the vector used is a plasmid, it can be transferred to the host cell by recombination.

1. IDENTIFICATION AND ISOLATION OF TRANSFORMED CELLS:

• The transformed cells are identified based on some selective property that has been acquired by the transformed cells. This selective property comes as a 'marker' along with the vector. Most frequently, markers coding for specific antibiotic resistance is used.
• The second employed method uses recipient cells with specific growth deficiencies (auxotroph) and vectors carrying genes thatovercome such deficiencies. Some traits exhibited by vector genes useful for identifying the transformed host cells (i.e., transformants) include:
• resistance against antibiotics, heavy metals
• production of antibiotics, bacteriocins, enterotoxins,
• metabolism/ degradation of aromatic compounds, sugars, hemoglobin
• Induction of plant tumor.

1. GENE CLONING:

Having introduced the rDNA and identified the transformed host cells, the next step is to produce many identical copies of the transformed cell, i.e.,, clones of the transformed cells.

1. EXPRESSION:

This involves transcription and translation of the information encoded by the desired gene using the host cell’s machinery.