Chapter 16: Recombinant DNA and Genetic Engineering

In today’s world, a genetic defect is generally considered to be a condition one must accept as permanent. However, there are those who believe our understanding of DNA will lead a time where ‘defective genes’ can be replaced with healthy genes. Such a transfer, known as gene therapy, is currently being tried in humans, and at least some results look promising. How is it possible to transfer genes from one organism to another? This is the field of recombinant DNA, which means genes from some organism other than one’s parents are inserted into an individuals genome.

Section 1: Toolkit for Recombinant DNA

Viruses function by inserting their own genetic material into the genome of a host animal, which then reads the viral genes and follows their instructions. Thus, viruses provide a natural case of recombinant DNA, one organism inserting their genes into another organism. In the age of genetic engineering, where humans decide which genes should be inserted into a given organism, we steal some of the tools used by these viruses. One of the most important tools for genetic engineering is a class of enzymes known as restriction enzymes. Restriction enzymes cut DNA at very specific sections of the DNA. When the DNA is cut, it is left with a sticky end, a single-stranded segment of DNA that will readily combine with a complimentary strand of DNA. Notice that if two segments of DNA have both been cut with the same restriction enzyme, they should have complimentary sticky ends. In such a case, an enzyme known as DNA ligase will come along and reconnect the segments of DNA (see page 256).

Why would two strands from different species combine to form a strand of recombinant DNA? Because the DNA molecule is universal. As it turns out, the molecule consists of two types of nitrogenous bases, Purines (A&G, the larger 2-ring structures) and Pyrimadines (T&C, the smaller 1-ring structures). For DNA to fit together, a purine must match with a pyrimadine.

Human insulin is made with such a process. The DNA segment for insulin has been inserted into the genome of a bacterium that is now used to produce excess insulin. But how do we get the instructions to make human genes? Remember, there are introns and exons in a eukaryotes genome, so copying DNA will result in the mistaken reading of ‘junk/spacer’ DNA. Therefore, human genes are read off a strand of mRNA (introns removed) by an enzyme known as reverse transcriptase, which runs through transcription in reverse (mRNA to single-stranded DNA to double-stranded DNA).

The use of recombinant DNA technology in the production of insulin has allowed for the production of large quantities of pure insulin because the bacteria serve as cloning vectors.

Section 2: PCR

At a crime scene, it is common to find just a drop of blood or small sample of saliva. How can useful DNA sample be extracted from such small samples? The process of polymerase chain reaction is required. In this process, an exceedingly small sample of DNA can be extracted and added to a beaker. The beaker is heated to 92 degrees Celsius, which causes the DNA molecule to uncoil and become single stranded. In the beaker, primers must be added. A primer is a segment of DNA that signals DNA polymerase to begin adding to a segment of DNA. Notice the unique situation here: we heat the DNA up to near boiling temperature and then expect an enzyme to catalyze the production of new DNA complimentary strands. Yet we know most enzymes denature at extreme temperatures. How can this work? The necessary enzyme was found in bacteria that live in the hot pools of Yellowstone National Park. The discovery of this protein turned out to be very lucrative for the researchers, as PCR requires the technology and the process is commonly used today. In the end, a few strands of DNA can be multiplied millions of times by running multiple PCRs.

Section 3: DNA Fingerprint – see the introduction to our lab

Section 4: How is DNA sequenced?

How can the sequence of DNA be read and deciphered? One method uses radiolabeled isotopes and gel electrophoresis. In this method, the four different nucleotides are each labeled with a unique radioisotope. When a nucleotide is labeled with a radioisotope it will have a unique structure because an additional atom is attached to the nucleotide (this is the atom that glows). Because DNA structure is based on the pairing of a purine and pyramadine, the extra atom throws off the pairing and the DNA molecule is disrupted. The last atom in the chain will be identifiable because it will glow the characteristic color of one of the four base nucleotides. By repeating this and then running the fragments through a gel electrophoresis procedure, researchers can see the last nucleotide that was added for a fragment of a given length, look at the next longest fragment and read the nucleotide that was inserted, and so on. This machine is called an automated gene sequencer.

Section 5: Isolating genes

How do researchers determine where a given gene occurs on a chromosome? A probe can be used. A probe is a molecule with a known segment of DNA attached to a radioisotope label.

Section 16.6 Using the Genetic Script

Of what use is figuring out the genome of an organism or knowing how to insert genes using restriction enzymes? The earliest application came about when researchers were able to genetically insert the DNA to make human insulin into the genome of E. coli bacteria. Prior to this technology, diabetic used the insulin extracted from cows or pigs. Today, Humalin, a brand name of insulin, supplies diabetics with human insulin made by bacteria. Other uses include mapping relationships of organisms (similar genotypes equate to close relations), forensics (the source of the last year’s anthrax bacteria were traced using DNA technology).

Section 16.7 Designer Plants

Source: An overview of the recombination process. Source: Novo - Nordisk promotional brochure,pg 6.

When researchers want to insert genes into a plant genome, they first remove the ‘tumor’ gene from the Ti plasmid (a small segment of DNA used by bacteria to transfer genes from one bacterium to another) from Agrobacterium tumefaciens. (The tumor-causing section is removed because this plasmid has been completely mapped and therefore it is known where that gene is found on the plasmid. By carefully selecting the restriction enzyme that is used, researchers can remove the specific gene that causes the tumor. In its place, researchers can insert a desired gene for glowing lucerase (figure 16.12), resistance to herbicide (figure 16.13) or many, many other options.

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