Recombinant DNA (rDNA): Technology, Plasmids, and Resources

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DNA Basics

DNA (or deoxyribonucleic acid) is the molecule that carries genetic information. It is part of a class of molecules commonly referred to as polynucleotides, given their long chains of nucleotides, and consists of two polynucleotide strands twisting around each other. Each strand stores information in the linear sequence of its nucleotides.

What is Recombinant DNA (rDNA)

Recombinant DNA (rDNA) is DNA created artificially by combining the DNA from two or more organisms into a single “recombinant” (recombined) molecule. The term “recombinant DNA technology” also commonly known as “DNA cloning,” “molecular cloning” and “gene cloning,” refers to the transfer of a segment of DNA from one organism to another organism (the “host cell”) where it reproduces. Recombinant DNA technology was first discovered in the 1970s and has been used in many academic fields, from medicine to agriculture. The process involves a sequence of steps as set forth below.

Steps in Recombinant DNA Technology

Step 1: Isolation of a gene of interest

First, recombinant DNA technology begins with isolation of a gene of interest, i.e., the donor DNA. To do this, a known DNA sequence from the donor cell is identified and removed with a restriction enzyme. When cut, each fragment of DNA has an overhanging piece of single-stranded DNA on its ends called “sticky ends” because they are able to attach to, and form base pairs with, any DNA molecule that contains a complementary sticky end. Restrictive enzymes are endonucleases that bacteria use in nature to defend against DNA viruses by cutting DNA at specific sites. In recombinant DNA technology, they are used to recognize and cut short, specific sequences of nucleotide bases in double-stranded DNA. Researchers have identified and isolated a number of different restriction enzymes differing in recognition sequences and other properties, which allow for cutting and joining DNA molecules from many different sources. For example, the enzyme EcoRI, from E.coli, cleaves to every site with the six-nucleotide sequence of GAATTC.

Step 2: Cloning vectors from the host cell are identified and removed

Second, cloning vectors from the host cell are identified and removed with the same restrictive enzyme applied to the donor DNA, resulting in a single-stranded DNA molecule with sticky ends complementary to those found on the fragmented donor DNA. Cloning vectors are DNA molecules in which another DNA fragment (i.e., foreign DNA) can be integrated and which are capable of independently replicating themselves and the foreign DNA once inserted into the host cell. The most commonly used cloning vector is the bacterial plasmid (a circular, double-stranded DNA sequence that replicates in bacteria). Other types include cosmids and yeast artificial chromosomes. The greatest variety of cloning vectors are cloning vectors developed for use in the bacterial host E.coli.

Step 3: The donor DNA is combined with the cloning vector to alter the genotype of the cloning vector

Third, the donor DNA is combined with the cloning vector to alter the genotype of the cloning vector, thereby creating “recombinant DNA.” To do so, the sticky ends of the donor DNA and the sticky ends of the cloning vector are lined up with each other, and the enzyme referred to as DNA ligase bonds the two pieces of DNA. The cloning vector contains a selectable marker that allows for identification of the recombinant DNA based on certain characteristics.

Step 4: The recombinant DNA is introduced into the host cell

Fourth, the recombinant DNA is introduced into the host cell in a process called “transformation.” Since foreign DNA (such as recombinant DNA) cannot easily penetrate a cell membrane, different methods, such as heat shock, are employed for transformation. When the host cell is non-bacterial, this process is called “non-bacterial transformation.” When the host cell is a phage, the process is called “transfection.”

Step 5: Upon being introduced into the host cell, the recombinant DNA replicates

Fifth, upon being introduced into the host cell, the recombinant DNA replicates, producing a large number of identical DNA molecules (referred to as “clones”). Such clones include the fragment of donor DNA originally linked to the cloning vector.

Step 6: Final Step

Finally, the cloned DNA segments can be recovered from the host cell and analyzed.

Resources about Recombinant DNA

The Basics of Recombinant DNA
In this problem set, you will learn about some of the basic techniques of recombinant DNA, and how recombinant DNA technology is applied to human health.
The Office of Biotechnology Activities: The Recombinant DNA Program promotes scientific advancement and safety in the conduct of basic and clinical recombinant DNA research.

Can you repair DNA by adding in other species’ DNA?

Cells undergo various types of damage through a typical day in the life of the organism. UV damage from the sun, viruses, industrial chemicals and radiation can cause broken chromosome and molecular lesions. This damage can interfere with the cell’s capability to pass down genetic information, either by changing the genome, resulting in mutations, or by preventing the transcribing of the gene. Cells are able to identify damage to their DNA and can correct some of this damage themselves. This depends on the environment surrounding the cell, the cell type, and how old the cell is. If, however, the cell is heavily damaged or is no longer able to repair itself, possible results are apoptosis (cell suicide), tumor or cell dormancy. In the case of apoptosis or cell dormancy, the cells are no longer able to divide, so any damage is confined to that cell and does not result in a cancerous tumor endangering the entire organism. This is similar to a defective electronic device: One would try to fix it, but if it was beyond repair, it would be kept turned off and not plugged in so it wouldn’t short the rest of the electricity in the house.

DNA damage, such as breaks in the single and double strands, can be detected by enzymes and repaired, as long as a “back up” (undamaged information) can be copied. On the other hand, since enzymes do not detect mutations, they can’t be repaired, and when a mutated cell divides and replicates itself, the mutation is also replicated. The DNA damage changes the structure of the helix, which is sensed by DNA repair molecules, which hurry to the site, attracting other molecules to join together and start working on the repair job. DNA ligase is one of the enzymes that specialize in repairing chromosomal damage.

If one is looking to repair DNA damage, the first resort should be having the cells themselves coming to the rescue. If the damage is too much for them to contend with, DNA from the same species should be enlisted.

Splicing together DNA from two different organisms is known as recombinant DNA technology, genetic engineering or bioengineering. By introducing new DNA, the organism isn’t being repaired; it is being altered. Genetically engineered crops are modified to provide them with certain traits or nutrients. An example of this would be golden rice, which is genetically engineered to include beta-carotene, or purple tomatoes, which have genes from the snapdragon flower. The oncomouse, a laboratory mouse produced by Harvard University, has been genetically modified to be highly susceptible to cancer, which makes it a favorite among cancer researchers.

In addition, genetic engineering is used commercially to provide diabetics with human insulin. Formerly, insulin was obtained from cows or pigs that had been killed, but the differences between animal insulin and human insulin sometimes resulted in side effects and immune reactions. Researchers removed the genes for human insulin and spliced them into plasmid, which was then implanted into bacteria, and when the bacteria multiplied, the human insulin multiplied as well.

In general, the field of genetic engineering has, in addition to its medical and scientific aspects, not a small amount of moral and emotional controversy too. Genetic engineering is restricted for some of these reasons, and all the legal aspects have not yet been completely clarified either.

Resources about DNA Repair

Cell, Heal Thyself: New Systems Biology Model Reveals How Cells Repair DNA Damage
Li Fan’s article about DNA replication and repair are two major biological processes essential for the maintenance of genomic stability and integrity.
New way to study how enzymes repair DNA damage

What do plasmids have to do with recombinant DNA cloning?

Recombinant DNA cloning refers to the process by which a segment of DNA (“donor DNA”) is identified and transferred from one organism (“donor cell”) to a host organism (“host cell”) where it is replicated, creating a population of identical cells, known as “clones.” The process as outlined above involves: (1) Identifying and removing donor DNA from a donor cell with a restriction enzyme, (2) identifying and removing a cloning vector from a host cell with the same restriction enzyme used to remove the donor DNA, (3) combining the donor DNA and the cloning vector to form recombinant (recombined) DNA and (4) introducing the recombinant DNA into the host cell, thus altering the genetic makeup of the host cell, via a process known as “transformation” (when the host cell is bacterial), “non-bacterial transformation” (when the host cell is nonbacterial) and “transfection” (when the host cell is phage). Once recombinant DNA is introduced into the host cell, it replicates, producing large numbers of identical DNA molecules that include the fragment of donor DNA originally linked to the cloning vector. Finally, the cloned DNA segments can be recovered from the host cell and analyzed.

What is the Cloning Vector?

A “cloning vector,” as used in recombinant DNA cloning, is a carrier DNA molecule in which a second DNA fragment can be integrated in such a way that the carrier molecule does not lose its capacity for self-replication and is used to introduce donor DNA into host cells.

What are the important Properties of Cloning Vectors?

Four important properties of all cloning vectors are the following:

1. They contain a DNA sequence allowing them to replicate themselves and donor DNA segments independently once inserted in a host cell.

2. They contain a number of unique restriction enzyme cleavage sites and can accommodate many different strands of donor DNA.

3. They carry a selectable and identifiable marker that researchers can use to distinguish host cells that carry cloning vectors from host cells that do not.

4. They are relatively easy to recover from a host cell.

Types of markers include antibiotic resistance markers and color selection markers. Types of cloning vectors include plasmids, cosmids and yeast artificial chromosomes, with genetically engineered plasmids being one of the most common.

What are Plasmids?

Plasmids are naturally occurring, small, circular, extra chromosomal double-stranded DNA molecules that contain an origin of replication and can replicate autonomously within bacterial cells. Plasmids are usually not required by their bacterial host cell for such host cell’s survival, but instead, carry genes that provide a selective advantage to the host cell, such as genes for resistance to antibiotics or heavy metals, or that produce antibiotics helping the host cell to compete for space or for food. Plasmids range in size from a few thousand base pairs to more than 100 kilobases, are found in numbers ranging from one per cell to hundreds per cell, and are easily recoverable from their host cell.

The greatest variety of cloning vectors used for recombinant DNA cloning are genetically engineered plasmids that have been developed for use in the bacterial host E.coli. Such plasmids successfully serve as cloning vectors because they do not contain much more than the essential elements required for the cloning process. These are: a DNA sequence allowing them to independently replicate themselves and the donor DNA segments once inserted in a host cell; a selectable and identifiable marker in the form of a drug-resistant gene; and a region in which DNA fragments from the donor cell can be inserted without interfering with the plasmids’ replication or effectiveness of the drug-resistant gene. Moreover, such plasmids can be genetically engineered to optimize their use as cloning vectors. For example, their circumference is often shortened to make them easier to work with.

Resources about Plasmids

Background information about plasmids that has information about: What is Recombinant DNA? What are Plasmids? What are Restriction Enzymes? How often does a Restriction Enzyme cut?

This website is part of an experiment in preserving the history of specific scientific areas. Some areas include Plasmids: Histories of a Concept.

SDSU.edu’sQuestions and answers about plasmids.

Resource by B.Sobey

I am a recent masters graduate from FL. I enjoy the beach, traveling, and writing. I just moved to South Carolina in hopes to find a job as a journalist.

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