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.

Double Helix Structure

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.

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.

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.

Recombinant DNA Quiz

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!


What you'll find

Recognition Sequences

Recognition Sequences for frequently used restriction endonucleases.

Synthesized Human Proteins

Information about human proteins that have been synthesized from eukaryotic and bacteria genes.

Gene Addition in Plants

Information about gene addition projects that have been done with plants.

Gene Subtraction in Plants

Information about gene subtraction projects that have been done with plants.

DNA Info

Basic information about what DNA is

DNA Replication

A SHOCKWAVE application illustrating DNA replication

Protein Synthesis

A video that illustrates protein synthesis

Gene Splicing

Information about how gene splicing differs from conventional agriculture

Gene Splicing

Information about the merits of agricultural gene splicing

Genetic Diseases

Information about treating genetic diseases in the womb

Gene Therapy

A Question and Answer about gene therapy

Recombinant DNA

The Recombinant DNA chapter of an online textbook

Recombinant DNA Technology

A Recombinant DNA problem set and tutorial

Recombinant DNA Research

The NIH Guidelines for research involving Recombinant DNA

Recombinant DNA Protocols

An online textbook covering the protocols for Recombinant DNA

Clinical Trials

A clearinghouse of links concerning Clinical Trials

Gene Therapy

Information about gene therapy for human patients

Insulin Synthesis

Recombinant DNA and the synthesis of human insulin

Medical Biotechnology

A repository of information concerning Medical Biotechnology


Created by Matthew Kuure-Kinsey and Beth McCooey for Biochemical Engineering Fall 2000