Recombinant DNA
technology refers to the techniques used to transplant genes from one
living source into another where it will be expressed.
How Does it work?:
The science of DNA technology includes the use of special
enzymes called restriction enzymes, DNA vectors, and the host organisms.
We will take a look at each of these groups in the following section.
Restriction Enzymes:
These special enzymes were discovered in the late 1960's
as naturally occurring agents in bacteria. They protect the bacterium against
foreign DNA from other organisms. Invading DNA is cut into pieces and made
inoperable. This process is called restriction.
As with any enzyme, these are specific in the job they do. Many of them
only recognize short, specific nucleotide sequences( recognition sequences)
and cut at specific points within those sequences. Bacteria protect their
own DNA by a process called methylation. During
this process methyl groups are added to the nucleotides within the recognition
sequence. There are several hundred restriction enzymes and about 100 different
recognition sequences.
Recognition Sequences: are symmetric in that the same
sequence of 4 to 8 nucleotides is found on both strands, but run in opposite
directions. The restriction enzymes usually cut the phosphodiester bonds
of both strands in a staggered manner. The result being both ends have
a single stranded area called the sticky ends.
It is within this space that the new piece of DNA is added, attaching to
the sticky ends. See the diagram below.
These unions are temporary until the enzyme DNA
ligase is added to catalyze the formation of the phosphodiester
bonds.
Vectors: are used as carriers for moving DNA from
test tubes into cells. Bacterial plasmids and viruses are the most widely
used vectors in DNA transfer. Bacterial cells can pick up the DNA through
the process of transformation. Lambda phages are used by eliminating the
middle of its liner genome and adding the foreign DNA in the created space.
The phage is then introduced into the bacterial cell where it replicated
itself via the lysogenic cycle.
Host Organisms: Bacteria are usually used as hosts
in genetic engineering. There are several reasons why they are chosen.
1. Bacterial DNA can be easily isolated and reintroduced into bacterial
cells. 2. Bacterial cultures grow quickly. Some disadvantages surface as
well: 1. Bacteria, being prokaryotic, may not be able to use the information
in eukaryotic genes. 2. Bacterial cells cannot make the the necessary changes
in transcription to produce some eukaryotic proteins. Eukaryotic cells
can also be used as hosts. Yeast cells and some plant and animal cells
can be a host for foreign DNA, but it is often difficult to get such cells
to take up engineered DNA.
Steps for using Bacteria and Plasmids to Clone Genes:
1. Isolation of two kinds of DNA.
2. Treatment of plasmid and foreign DNA with the same restriction
enzyme.
3. Mixture of foreign DNA with clipped plasmids.
4. Addition of DNA ligase.
5. Introduction of recombinant plasmid into bacterial cells.
6. Production of multiple gene copies by gene cloning and
selection process for transformed cells.
7. Final screening for transformed cells.
Additional Methods for Analyzing and Cloning Nucleotide Sequences:
1. Gel Electrophoresis. is used
to separate either nucleic acids or proteins based upon molecular size,
charge and other physical properties. Various DNA's can be identified by
their characteristic banding patterns after being cut with their particular
retraction enzymes. Genes can be isolated, purified and recovered from
the gel with full biological activity.
2. DNA Synthesis. It is possible
to create known short DNA sequences in the laboratory. The Sanger
Method is used to identify the sequence of a DNA molecule.
This method is based on: a). use of restriction enzymes into small reproducible
fragments. b). produce in a test tube DNA strands complimentary to a strand
from the restricted fragment. c). Add a modified nucleotide that blocks
further DNA synthesis.
3. The Polymerase Chain Reaction.
This is another process that will allow DNA to be copied and amss produced
in vitro. This process needs only a small bit of DNA and is being used
in reconstruction of ancient DNA, minute specimens from crime secenes,
DNA from cells infected with hard to find diseases, and DNA from single
embryonic cells for prenatal diagnosis.
4. Hybridization. is used to
determine the presence of a specific nucleotide sequence. Labeled probes
complementary to the gene of intrest are allowed to bind to DNA from the
cells being tested. This will determine if the gene being sought is present.
It will also account for the numbers of these sequences, the size of the
restriction fragment, and determine if the gene is made of mRNA, and how
much of the mRNA is present.
Much of the above technology is being practiced in The Human
Genome Project. This is an enormous effort to map and sequence the DNA
of the human genome. Click here
for a link to this project.
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