Gene manipulation is a technique that involves manipulation of genetic material, also termed as Genetic Engineering. The technology is basically used by scientists to enhance or modify the characteristics of an individual organism. The first genetically modified organism to be created was a bacterium, in 1973 and in 1974, the same techniques were applied to mice. Since then, genetic modification has advanced largely with its applications in countless fields.
The process of gene manipulation involves isolation of genetic material from an organism (plant or animal); transfer of genetic material to a suitable vector; transferring of a vector to a host through transfection or transformation.
Table of Contents
Various Tools and Techniques For Genetic Modification
Gene manipulation can be carried out through various tools and techniques.
1. DNA/RNA extraction
The DNA/RNA is isolated and extraction from cells, this can be done by breaking open the cells using enzymes to destroy macromolecules that are not needed. The cells are broken using a ‘lysis buffer’, and the macromolecules inactivated using enzymes such as Proteases for RNA, proteins. Precipitation of DNA done using alcohol. DNA appears to be in white mass/gelatinous form.
2. PCR: Polymerase Chain Reaction
The techniques that amplify a single segment of DNA into a thousand copies within a short period. Desired DNA amplified through recurrent replication process. In a PCR machine a target DNA with primer up to 20-30 bp, a buffer solution, a free DNA with nucleotide (d ATP, d GTP, d CTP, d TTP) with a reagent MgCl2/KCl along with DNA polymerase enzyme (TAQ enzymes) is kept. Millions of copies of the DNA can be obtained in 25 cycles.
Restriction Endonucleases: They are enzymes that can recognize sites in DNA and cut it at specific motifs. For example, Eco RI obtained from strain R, Bam II etc.
DNA Ligase: This enzyme joins two DNA length together forming phosphodiester bond from 5’ phosphate end of a chain to 3’ end of another chain. The main function involves repairing “nicks” and breaks in single DNA strands.
DNA Polymerase: It makes DNA from nucleotides and plays an important role in DNA replication.
4. Gel Electrophoresis
Gel electrophoresis is the technique that separates molecules according to their size using charge in the electric field. The nucleic acids that are negatively charged are loaded at the negative electrode in the gel and pulled towards positive electrode when an electric field is applied. Smaller molecules move faster than the larger molecules and get separated in the gel on the basis of their size forming bands that are observed using fluorescent dyes.
5. Hybridization, Southern and Northern Blotting
The sample DNA/RNA is fragmented and are separated on a gel and then transferred to Nylon membrane. They are then incubated with probes complementary to the sequence of interest. Southern blotting is a technique used for DNA and Northern blotting is used for RNA.
It is “a vehicle” in genetic engineering for transfer of foreign genetic material into another cell. Types of vectors include Plasmids, cosmids, bacteriophage, artificial chromosomes etc. They are themselves DNA molecules.
7. Molecular Cloning
Involves use of Recombinant DNA molecule for replication within the host organism. Small cloned fragments can be used for the study of specific genes in gene manipulation. Steps involved choosing vector and host organism, preparation of specific DNA to be cloned, creating Recombinant DNA and introducing it into the host; finally screening of suitable clones with the gene of interest. The most common screening method is “Blue-white screening” used in identifying reporter proteins like X-gal/LacZ gene forming blue-white colonies allowing proper selection of recombinant ones.
8. The three T’s: TRANSDUCTION, TRANSFECTION, AND TRANSFORMATION
• Transduction: foreign DNA introduced in a cell by a viral vector. For example, Bacteriophage infecting bacterial cells. Also describes the introduction of a recombinant viral vector into target cells.
• Transfection: A forced introduction of small molecules such as DNA, RNA into eukaryotic cells. Common transfection methods include Electroporation, Calcium phosphate, gene gun, microinjection etc.
• Transformation: DNA uptake by the bacterial cells from the environment. Recombinant plasmid DNA can also be introduced into the competent bacterial cells that take up extracellular DNA from the environment.
The listed methods for gene manipulation can be applied to in-vivo and in-vitro techniques. The ability to clone a DNA can have far consequences in future with various pros and cons. It has become possible to identify new DNA sequences along with new potential genes that be easily cloned using any above-listed methods for a new genetic product. It has advanced in the field of Recombinant DNA technology that has made the availability of certain therapeutic medicines, treatment of any genetically related diseases, modification of animal and plant genes for healthier disease resistance organism.