Recombinant DNA

This process creates a new strand of DNA through the combination of both strands, like how sexual intercourse creates a child through the combination of egg and sperm from the parents.

Recombinant DNA itself is a unique strand of DNA that comes from the combination of two “parent” DNA strands. This strand cannot be found naturally in organisms. While crossing over during meiosis can produce recombined DNA, the term recombinant DNA is used for the strands produced by scientists through splicing (Recombinant DNA).

Recombinant DNA originates in the mid 20th century. A scientist named Arthur Kornberg had originally copied DNA, which showed that cloning was a feasible adventure. Afterwards, a Swiss biochemist named Werner Arber discovered restriction enzymes in bacteria. These enzymes defend the bacteria by attacking the DNA of viruses while ignoring DNA in the bacteria itself. Arber used these enzymes to cut DNA molecules, and then used an enzyme called ligase to re-bond DNA molecules together. With these two discoveries, recombinant DNA went from a fantasy into a reality. (Carroll)

In 1972, the first recombinant DNA molecules were created by Paul Berg of Stanford University. He combined the DNA of two different organisms. He used a restriction enzyme to isolate a gene from a carcinogen monkey virus. He then used ligase to join the section of the monkey virus DNA with molecule of DNA from a bacterium to make the first recombinant DNA molecule (Carroll).

In 1973, Stanley Cohen and Herbert Boyer worked together to create the first recombinant DNA organism using the techniques that were developed by Paul Berg. This experiment became to basis of today’s genetic engineering. Stanley Cohen had found a way to extract the plasmid of a cell, a DNA molecule separate from chromosomal DNA, and implant it in another cell. Boyer had used restriction enzymes to remove specific sections from a strand of DNA. They combined their research and made the first recombinant DNA organisms (Carroll).

First they removed the plasmids from the cytoplasm of a cell. Then, using restriction enzymes, they cut the DNA at specific locations and recombined the different strands. Finally, they inserted the new, recombined DNA into E. coli bacteria cells. The E. coli divided, passing down the changed strands. These bacteria cells then produced specific proteins that could be used by humans. Today, the process of recombinant DNA remains unchanged. Scientists still use restriction enzymes and bacteria cells to divide and carry the recombinant DNA (Carroll).

However, this new technology was controversial back then. At the Asilomar Conference in 1975, the leading recombinant DNA specialists agreed to continue their research with caution, as infected viruses could spread and cause harm to humans and the environment. There, scientists identified and evaluated the risks of recombinant DNA. After the conference, recombinant DNA was allowed to flourish and prosper (Berg).

But how exactly does one go about recombining DNA strands? There are two methods one could use: transformation and non- bacterial transformation. Transformation is when a piece of DNA is chosen to be inserted into a vector. This DNA is altered in a laboratory to include things that make it easier to isolate and analyze cloned DNA. These things include markers to show the origin of replication, restriction sites, and genes that let researchers select cells transformed by recombinant plasmids.

When the DNA is inserted into a bacterium, this bacterium is exposed to an environment that doesn’t allow untransformed cells to grow. For example, if a gene gives resistance to a substance such as tetracycline, when inserted into a vector, the vector will grow in an environment with tetracycline present (Solomon, Berg, Martin).

After a specific gene is chosen, scientists have to cut it out of the strand. They do this with restriction enzymes. There are many different types of restriction enzymes, each with their own characteristics. They all cut a DNA strand at a specific place. For example, the restriction enzyme HindIII cuts a DNA strand at AAGCTT, while EcoRI cuts a DNA strand at GAATTC. Bacteria produce restriction enzymes as a defense mechanism.

When a bacteriophage, a virus that attacks a bacterium, injects its DNA into a bacterium, the bacteria defends itself with the restriction enzymes. The enzymes attack the bacteriophage’s DNA. In order to protect its own DNA, the cell modifies it so that the enzyme doesn’t recognize it as a bacteriophage’s DNA (Solomon, Berg, Martin).

After the gene from the target strand is cut, the vector, a substance that is used to deliver the gene into a bacteria cell, is then subjected to the same restriction enzyme. The enzyme cuts the DNA of the vector at the same place as the gene was. This makes it so that when the two DNA is mixed, the nitrogen bases match up evenly with adenine matching up with thymine and cytosine matching up with guanine (Solomon, Berg, Martin).

When the bacteriophage DNA is sliced, it goes from a circular form to a linear form. The segment of DNA with the gene for recombination is set in the bacteriophage DNA, and is then bonded by an enzyme called ligase. When the final DNA is formed, it is placed inside a bacterium or other vector. The bacterium or vector then either divides through mitosis, passing on the changed DNA to future generations, or infects a cell, which then divides through mitosis itself. Either way, something divides and passes on the changed DNA to a future generation (Solomon, Berg, Martin).