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. 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! The URL
What you'll find
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.
Basic information about what DNA is
A SHOCKWAVE application illustrating DNA replication
A video that illustrates protein synthesis
Information about how gene splicing differs from conventional agriculture
Information about the merits of agricultural gene splicing
Information about treating genetic diseases in the womb
A Question and Answer about gene therapy
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
A clearinghouse of links concerning Clinical Trials
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