About Recombinant DNA

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What is recombinant DNA?

How does recombinant DNA work?

A Closer Look

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What is recombinant DNA?

Deoxyribonucleic acid, or DNA, is the blueprint for life. Inside every cell in your body, DNA contains the code that determines who you are and what traits you have. Recombinant DNA is DNA from two different sources that has been combined in vitro (outside living organisms). There are three main reasons for creating recombinant DNA: (i) to create a protein product, (ii) to create multiple copies of genes, and (iii) to insert foreign genes into other organisms to give those organisms a new trait (Campbell & Reece, 2002).

Recombinant DNA is used widely today to create large amounts of protein for treating certain illnesses. In 1982, insulin became the first recombinant DNA drug to hit the market (NHGRI, 2003). A person with diabetes does not produce adequate insulin. Insulin, a protein, can now be produced in large quantities by bacteria that have been given the human insulin gene (Hormones, n.d.; Stanford University, 2002; G. Stein & J. Stein, 2002). Another example of a protein that is made by bacteria for medical use is human growth hormone (Hanna, 2004; G. Stein & J. Stein, 2002).

The creation of multiple copies of a gene is valuable for genetic research. The availability of multiple copies of a gene has many advantages including the determination of the nucleotide sequence of a gene (Campbell & Reece, 2002).

Inserting genes that originated in one organism into another organism is proving indispensable in agriculture and other fields. In agriculture, adding genes to plants to make them draught or insect resistant is already common practice (Anunson et al, 1999; Campbell & Reece, 2002). Another use is the creation of bacteria that will help clean up toxic waste. A bacteria has been created using recombinant technology that can digest oil from an oil spill (Campbell & Reece, 2002).

How does recombinant DNA work?

Here is an overview of how a gene from an organism can be inserted into a bacterium. First, the gene of interest must be identified. For example, the insulin gene would have to be localized in the human genome. Then a plasmid has to be isolated from bacteria cells. A plasmid is a circular, double-stranded DNA sequence that replicates in bacteria and is separate from the bacterial chromosome. The gene is inserted into the plasmid, and the plasmid is taken up by a bacterium. The bacteria reproduce, and start creating the desired protein (Campbell & Reece, 2002).

A illustration showing showing how human insulin can be produced by bacteria using recombinant DNA (MIT, 1989).

 

Bacterial Plasmids (Kimball, 2004).

Restriction enzymes, discovered in 1968, are important parts of this process (NHGRI, 2003). In nature, restriction enzymes are a bacterium’s self-defense. A restriction enzyme cuts in between a certain sequence of nucleotides, called the restriction sight, which is 4-8 nucleotides long. Every time that sequence occurs in the bacterium’s own DNA, methyl groups (-CH3) are added to adenines or cytosines which prevent the restriction enzyme from working. Any time foreign DNA, such as a phage (a bacterial virus), enters the bacterium, the bacterium’s restriction enzyme would cut the phage’s DNA into pieces. Although not all bacteria have restriction enzymes, there are wide varieties of restriction enzymes that have been discovered and continue to be discovered (Campbell & Reece, 2002).

Restriction enzymes are used to cut open a plasmid and the same enzyme is used to cut the desired gene out of the chromosome. This makes two matching cuts, and when the gene and plasmid are combined, they form a temporary bond. Another enzyme, DNA ligase, is used to create a permanent seal (Campbell & Reece, 2002).

 

An example of how a restriction enzyme might work. The restriction site is g-g-a-t-c-c (Kimball, 2004).

 

This is an example of using restriction enzymes to insert DNA into a plasmid. The restriction site is g-a-a-t-t-c (MIT, 1989).

A Closer Look

To simplify things, the foregoing has described recombinant DNA in terms of one cell, one restriction enzyme, etc. When scientists are attempting to make recombinant DNA, it is done on a much larger scale. Millions of plasmids are mixed with millions of genes. Millions of restriction enzymes are dumped in to make millions of cuts. Plasmids bind to plasmids, plasmids bind to genes, genes bind to genes, plasmids bind to multiple genes, and the whole thing is a mess (Campbell & Reece, 2002).

To solve this problem, a cool trick is used. The starting plasmids (the ones that are going to be modified) are called cloning vectors, which is a molecule of DNA that can carry foreign DNA into a cell and replicate there. These plasmids include a gene that confers resistance to ampicillin. They also contain a second gene (for example lacZ). The restriction site, where the restriction enzyme will make a cut, is in the lacZ gene. If foreign DNA is inserted into the lacZ gene where the restriction enzyme made its cut, the lacZ gene will no longer work. The restriction enzymes are mixed with the plasmids, and then the desired genes, the genes we want to combine with the plasmids, are added. Then the bacteria are induced to take up the plasmids. Changes in heat, or chemicals that are added, are ways to make bacteria do this. After the bacteria are induced to take up the plasmids, the bacteria are grown on a medium of ampicillin and a substance that reacts to the protein product of lacZ. A bacteria colony that took up a plasmid with an intact lacZ gene, meaning that it is not a recombinant plasmid, will turn blue. Bacteria that did not take up any plasmids will not be able to grow on the ampicillin. Therefore, only bacteria that took up a plasmid will be growing, and only bacteria that had something inserted into the lacZ gene will be white (Campbell & Reece, 2002).

The next step is determining which of the remaining non-blue bacteria took up the right gene. We know with certainty that they took up some DNA but we do not know if it was the correct DNA. Scientists can look for the desired protein product (insulin for example) or they can look for the gene. To look for a gene, a sequence of nucleotides that occur in the gene must be known. A complementary sequence of DNA or RNA is created to the known sequence in the gene. This is called a nucleic acid probe. The probe is labeled with a radioactive tag or with a fluorescent tag so that it can be found. The bacteria’s DNA is denaturized, meaning the DNA is unraveled. This can be done with heat or chemicals. When the tag is added to the bacteria it will bond with complementary DNA (the DNA that is part of the gene we are looking for) inside a bacterium. We then know which bacterium has the desired gene and it can be grown in large tanks to produce the desired protein. This process of using a nucleic acid probe is known as nucleic acid hyperbridization (Campbell & Reece, 2002).