The Basics of Recombinant DNA
So What Is rDNA?
That's a very good question! rDNA stands
for recombinant DNA. Before
we get to the "r" part, we need to understand DNA. Those of you with
a background in biology probably know about DNA, but a lot of ChemE's haven't
seen DNA since high school biology. DNA is the keeper of the all the
information
needed to recreate an organism. All DNA is made up of a base consisting
of sugar, phosphate and one nitrogen base. There are four nitrogen bases,
adenine (A), thymine (T), guanine (G), and cytosine (C). The nitrogen
bases are found in pairs, with A & T and G & C paired together. The
sequence
of the nitrogen bases
can be arranged in an infinite ways, and their structure is known as
the famous "double
helix" which is shown in the
image below. The sugar used in
DNA is deoxyribose. The
four nitrogen bases are the same for all organisms. The
sequence and number of
bases is what creates diversity. DNA does not
actually make the
organism, it only makes proteins.
The DNA is transcribed
into mRNA and mRNA is
translated into protein, and the protein
then forms the
organism. By
changing the DNA sequence, the way
in which the protein is
formed changes. This
leads to either a different protein, or an inactive protein.
Now that we know what DNA is, this is where the recombinant comes in.
Recombinant DNA is the general name for taking a piece of one DNA, and
and combining it with another strand of DNA. Thus, the name recombinant!
Recombinant DNA is also sometimes referred to as "chimera." By
combining two or
more different strands of DNA, scientists are able to create a new strand of
DNA.
The most common recombinant process involves combining the DNA of two
different organisms.
How is Recombinant DNA made?
There are three different methods by which
Recombinant DNA is made. They are
Transformation, Phage Introduction, and Non-Bacterial Transformation. Each
are described separately below.
Transformation
The first step in transformation is to select a piece of DNA to be inserted
into a vector. The second step is to cut that piece of DNA with a restriction
enzyme and then ligate the DNA insert into the vector with DNA Ligase. The
insert contains a selectable
marker which allows for identification of recombinant molecules. An antibiotic
marker is often used so a host cell without a vector dies when exposed to a
certain
antibiotic, and the host with the vector will live because it is resistant.
The vector is inserted into a host cell, in a process called transformation.
One
example of a possible host cell is E. Coli. The host cells must be specially
prepared to take up the foreign DNA.
Selectable markers can be for antibiotic resistance, color changes, or any
other
characteristic which can distinguish transformed hosts from untransformed
hosts.
Different vectors have different properties to make them suitable to different
applications. Some properties can include symmetrical cloning sites, size, and
high copy number.
Non-Bacterial Transformation
This is a process very similar to Transformation, which was described above.
The
only difference between the two is non-bacterial does not use bacteria such as
E. Coli
for the host.
In microinjection, the DNA is injected directly into the nucleus of the cell
being
transformed. In biolistics, the host cells are bombarded with high velocity
microprojectiles, such as particles of gold or tungsten that have been coated
with DNA.
Phage Introduction
Phage introduction is the process of transfection, which is equivalent to
transformation,
except a phage is used instead of bacteria. In vitro packagings of a vector is
used.
This uses lambda or MI3 phages to produce phage plaques which contain
recombinants.
The recombinants that are created can be identified by differences in the
recombinants and non-recombinants using various selection methods.
How does rDNA work?
Recombinant DNA works when the host cell
expresses protein from the recombinant genes.
A significant amount of
recombinant protein will not be produced by the host unless expression
factors are added. Protein expression depends upon the gene being surrounded by
a collection of signals which provide instructions for the transcription and
translation
of the gene by the cell. These signals include the promoter, the ribosome
binding
site, and the terminator. Expression vectors, in which the foreign DNA is
inserted,
contain these signals. Signals are species specific. In the case of E. Coli, these
signals must be E. Coli
signals as E. Coli is unlikely to understand the signals of
human promoters and
terminators.
Problems are encountered if the gene contains introns or contains signals which
act
as terminators to a bacterial host. This results in premature termination, and
the recombinant
protein may not be processed correctly, be folded correctly, or may even be
degraded.
Production of recombinant proteins in eukaryotic systems generally takes place
in
yeast and filamentous fungi. The use of animal cells is difficult due to the
fact
that many need a solid support surface, unlike bacteria, and have complex
growth
needs. However, some proteins are too complex to be produced in bacterium,
so eukaryotic cells must
be used.
Why is rDNA important?
Recombinant DNA has been gaining in
importance over the last few years, and
recombinant DNA will only become more important in the 21st century as genetic
diseases become more
prevelant and agricultural area is reduced. Below are
some of the areas where
Recombinant DNA will have an impact.
- Better Crops (drought & heat resistance)
- Recombinant Vaccines (ie. Hepatitis B)
- Prevention and cure of sickle cell anemia
- Prevention and cure of cystic fibrosis
- Production of clotting factors
- Production of insulin
- Production of recombinant pharmaceuticals
- Plants that produce their own insecticides
- Germ line and somatic gene therapy
What does the future hold?
Now that we've figured out the basics
behind what Recombinant DNA are, it's
time to look at how Recombinant DNA will impact the future. Which industries
and fields will be shaped by rDNA? How will rDNA effect the health and
lifestyles of RPI students in the next generation? Click over to our
rDNA Impact Statement
to find out the answer!
Pop Quiz Time!
To help you determine how well you know
Recombinant DNA, we
have generously decided to provide you with a basic quiz that even a
senior ChemE should be able to do. Be sure and look over the additional
information provided below, because these questions could be tricky! All
the information needed to answer the questions can be found on this page,
or the associated pages. When you're ready, click below.
Additional Information
The information presented above is only an
introduction to the wonders of
Recombinant DNA. In order to fulfill your desire for knowledge, Matt and
Beth have scoured the web for the best websites with in-depth knowledge
concerning rDNA. You will find the links below and a brief
description of what the page describes. Enjoy!
|
The URL |
What you'll find |
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Recognition Sequences for frequently used restriction endonucleases. |
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Information about human proteins that have been synthesized from eukaryotic and bacteria genes. |
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Information about gene addition projects that have been done with plants. |
|
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Information about gene subtraction projects that have been done with plants. |
|
|
Basic information about what DNA is |
|
|
A SHOCKWAVE application illustrating DNA replication |
|
|
A video that illustrates protein synthesis |
|
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Information about how gene splicing differs from conventional agriculture |
|
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Information about the merits of agricultural gene splicing |
|
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Information about treating genetic diseases in the womb |
|
|
A Question and Answer about gene therapy |
|
|
The Recombinant DNA chapter of an online textbook |
|
|
A Recombinant DNA problem set and tutorial |
|
|
The NIH Guidelines for research involving Recombinant DNA |
|
|
An online textbook covering the protocols for Recombinant DNA |
|
|
A clearinghouse of links concerning Clinical Trials |
|
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Information about gene therapy for human patients |
|
|
Recombinant DNA and the synthesis of human insulin |
|
|
A repository of information concerning Medical Biotechnology |
Created by Matthew Kuure-Kinsey and Beth McCooey for Biochemical Engineering Fall 2000