Tools and Methods in Genetic Engineering
Genetic engineering
-is the direct modification of an organism’s genome, which is the list of specific
traits (genes) stored in the DNA.
-Changing the genome enables engineers to give desirable properties to
different organisms.
Genetically Modified Organisms (GMOs)- Organisms created by genetic
engineering
-All genetic changes affect the protein synthesis of the organism.
-By changing which proteins are produced, genetic engineers can affect the
overall traits of the organism.
Different methods of Genetic Modification:
• Inserting new genetic material randomly or in targeted locations
• Direct replacement of genes (recombination)
• Removal of genes
• Mutation of existing genes
Bacteria
-are the most common GMOs because their simple structure permits easy
manipulation of their DNA.
- One of the most interesting uses for genetically modified bacteria is the
production of hydrocarbons (plastics and fuels) usually found in fossil fuels.
Plants
-being the primary source of food are also one of the subjects for modification.
-Plants are genetically modified to become insect, herbicide, drought or freeze
and disease resistant, higher yield, faster growth, improved nutrition, and longer
shelf life.
Endangered Animals
- are now getting extinct but to consider, genetic engineering can be a solution
to restore their genes.
-Genetic engineers were able modify animals as to protein tracking, disease
detection using bioluminescent imaging (BLI) to identify different types of cells
and creating novelty pets such Glofish.
Tools and Methods of Genetic Engineering
1. Transgenic Organisms
• Not all cells will take up DNA during transformation.
• It is essential to identify the cells that undergo a transformation and those that
have not.
• Generally, plasmids carry genes for antibiotic resistance, and transgenic cells
can be
selected based on the expression of those genes and their ability to grow on
media
containing that antibiotic.
• Alternative methods of selection depend on the presence of other reporter
proteins such as
the x-gal/lacZ system, or green fluorescence protein, which allow selection
based on color
and fluorescence, respectively.
3. Selection of Small Self-Replicating DNA
• In biotechnology, once the gene of interest has been amplified and both the
gene and plasmid are cut by restriction enzymes, they are ligated together,
generating what is known as a recombinant DNA.
• Viral (bacteriophage) DNA can also be used as a vector, as can cosmids,
recombinant plasmids containing bacteriophage genes.
4. Eukaryotic Host
• Eukaryotic organisms are preferred to produce human proteins since these hosts
with complex structures (with distinct organelles) are more suitable to synthesize
complex proteins.
• The commonly used eukaryotic organism is the yeast, Saccharomyces
cerevisiae. It is a non-pathogenic organism routinely used in the brewing and
baking industry.
5. Prokaryotic Host
• Escherichia coli was the first organism used in the DNA technology experiments
and continues to be the host of choice by many workers. Under a suitable
environment, the number of E. coli can double every 20 minutes.
• As the bacteria multiply, their plasmids (along with foreign DNA) also multiply to
produce millions of copies, referred to as a colony or in a short clone.
• The term ‘clone’ broadly refers to a mass of cells, organisms, or genes that
results from the multiplication of a single cell, organism, or gene.
6. Polymerase Chain Reaction
• PCR is known as the polymerase chain reaction.
• It is efficient because it multiplies the DNA exponentially for each of the 25 to 75
cycles. A cycle takes only a minute, and each new segment of DNA that is made
can serve as a template for new ones
7. Restriction Enzymes (Molecular Scissor)
• The discovery of enzymes known as restriction endonucleases has been essential
to protein engineering.
• These enzymes cut DNA at specific locations based on the nucleotide sequence.
• Hundreds of different restriction enzymes, capable of cutting DNA at a distinct
site, have been isolated from many different strains of bacteria.
• DNA cut with a restriction enzyme produces many smaller fragments of varying
sizes.
• These can be separated using gel electrophoresis or chromatography.
8. Gel Electrophoresis
• Purifying DNA from cell culture, or cutting it using restriction enzymes would not
be of much use if we could not visualize the DNA – that is, find a way to view
whether your extract contains anything, or what size fragments you have cut it
into.
• One way to do this is by gel electrophoresis where gels are used for a variety of
purposes, from viewing cut DNA to detecting DNA inserts and knockouts.
9. DNA Ligase
• It is often necessary to link two or more individual strands of DNA, to create a
recombinant strand, or close a circular strand that has been cut with restriction
enzymes.
• Enzymes called DNA ligases can create covalent bonds between nucleotide
chains.
• The enzymes DNA polymerase I and polynucleotide kinase are also important in
this process, for filling in gaps, or phosphorylating the 5′ ends, respectively.
10. Polymerases
• The groups of enzymes that catalyze the synthesis of nucleic acid molecules are
collectively referred to as polymerases. It is customary to use the name of the
nucleic acid template on which the polymerase acts.
