Gene Cloning and Biotechnology
Biotechnology : Using Genetic Engineering to alter genes of bacteria,
plants and animals (humans)
Gene Therapy is one example of biotechnology. The goal is to alter the
phenotype in a human, by altering their genetic makeup.
Ex. Child suffering from SCID, now has proper B and T lymphocytes
with the proper gene placed into her stem cells.
When genetic engineers alter genes of organisms other than humans,
the new organism is said to be Transgenic.
Ex. Soybean plants can be made resistant to herbicides so farmers can
kill weeds without damaging their crops.
- One of the most common practices in Biotechnology is "Gene
Cloning"
There are two main ways to clone (copy) a gene:
A) Using recombinant DNA (rDNA)
B) Using Polymerase Chain Reaction (PCR)
A) Using rDNA
Definition: DNA having genes from two different organisms, often
produced in the laboratory by introducing foreign genes into a bacterial
plasmid.
- A vector must be used to introduce the
Recombinant DNA. A plasmid is the most common vector (they are
small accessory rings of DNA founds in bacteria)
- The plasmid has to be removed from the bacteria and then a foreign
gene is inserted into it as follows:
1. Firstly a specific restriction enzyme is used to break the plasmid DNA
open as well as to break the host's DNA (containing the desired gene).
This restriction enzyme will cleave the DNA at a specific pattern of
bases.
Ex. One restriction enzyme will cleave DNA only at the following site:
AGAATTCGC
TCTTAAGCG
- After cleavage, both the host's DNA and the Plasmid DNA have
complimentary sticky ends.
- The new foreign DNA can now be joined into the plasmid DNA with
the help of a specific enzyme called DNA Ligase. The enzyme, Ligase,
then acts like a glue and seals the foreign DNA to the plasmid DNA.
- This new recombined DNA is then put back into the bacteria. As the
bacteria asexually reproduces, the rDNA will replicate itself so that
every new bacterium will contain a copy of this newly inserted foreign
gene. Eventually there will be many copies of this foreign gene.
Illustrating rDNA formation and use:
- A more detailed view:
- For the bacterial cells to be capable of making desired protein from
that desired gene (DNA). The DNA must be pre-processed into (cDNA)
"Complimentary DNA" before it was actually joined to the vector.
- Remember that during transcription DNA
mRNA, but before
the mRNA can be translated it must have specific Ribozymes (NOT
Ribosomes) cut out the useless introns and save the meaningful exons
for translation.
- The problem is that the bacteria do not have those proper ribozymes
necessary for processing. To allow for the bacterial cell to use the
desired gene, the desired DNA (gene) must be made so that it does not
contain any introns. This is possible in the lab with the help of a special
enzyme called "Reverse Transcriptase". Reverse transcriptase can use
the processed mature mRNA to make a piece of DNA that is perfectly
complementary to this mRNA. Now this cDNA (complimentary) has
been made and it is ready to be joined to the vector.
- Viral DNA can also be used as a vector to carry recombinant DNA
into a cell. When a virus containing recombinant DNA infects a cell, the
viral DNA enters. Inside the host cell it can direct the reproduction of
many more viruses.
Viral Vector vs. Bacterial Plasmid Vector:
- Viral vectors are also used to create genomic libraries. A genomic
library is a collection of engineered viruses that carry all the genes of a
species. It takes about 10 million viruses to carry all the genes of a
mouse.
B) Using PCR
- During Polymerase Chain Reaction, a single piece of DNA (gene) can
be copied millions of times without having to use another organism.
- In a lab setting, a the desired gene is placed into a solution containing
the following:
1. A large supply of DNA nucleotides
2. A heat-resistant DNA Polymerase
3. Primers - small starter segments of DNA that compliment an outside
piece of the gene.
- The procedure is illustrated below.
Benefits of Gene Cloning and Transgenic Organisms:
- Often the desired genes make useful proteins (like vaccines,
antibodies, enzymes etc.), giving rise to countless benefits.
Ex. Protein hormones like insulin can by produced using yeast
cells.
- Interferon is a protein used in cancer treatments; it acts to help the
immune system. Interferon can be mass-produced using bacteria.
- Producing new strains of livestock and crops:
Ex. Frost-Hardy plants: