- 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
- 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
- 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
- 4. Addition of DNA ligase.
- 5. Introduction of recombinant plasmid into
- 6. Production of multiple gene copies by gene
cloning and selection process for transformed cells.
- 7. Final screening for transformed
- Additional Methods for Analyzing and Cloning Nucleotide
- 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
- 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.
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
- 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
for a link to this project. Genome
- To practice this type of technology click here
to link up with AP
Lab Number 6 Molecular Biology.