Basic Techniques to Manipulate Genetic Material (DNA and RNA)

To understand the basic techniques used to work with nucleic acids, remember that nucleic acids are macromolecules made of nucleotides (a sugar, a phosphate, and a nitrogenous base) linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge. An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. Exposure to high temperatures (DNA denaturation) can separate the two strands and cooling can reanneal them. The DNA polymerase enzyme can replicate the DNA. Unlike DNA, which is located in the eukaryotic cells’ nucleus, RNA molecules leave the nucleus. The most common type of RNA that researchers analyze is the messenger RNA (mRNA) because it represents the protein-coding genes that are actively expressed. However, RNA molecules present some other challenges to analysis, as they are often less stable than DNA.

DNA and RNA Extraction

To study or manipulate nucleic acids, one must first isolate or extract the DNA or RNA from the cells. Researchers use various techniques to extract different types of DNA (Figure 2). Most nucleic acid extraction techniques involve steps to break open the cell and use enzymatic reactions to destroy all macromolecules that are not desired (such as unwanted molecule degradation and separation from the DNA sample). A lysis buffer (a solution which is mostly a detergent) breaks cells. Note that lysis means “to split”. These enzymes break apart lipid molecules in the cell membranes and nuclear membranes. Enzymes such as proteases that break down proteins inactivate macromolecules, and ribonucleases (RNAses) that break down RNA. Using alcohol precipitates the DNA. Human genomic DNA is usually visible as a gelatinous, white mass. One can store the DNA samples frozen at –80°C for several years.

Scientists perform RNA analysis to study gene expression patterns in cells. RNA is naturally very unstable because RNAses are commonly present in nature and very difficult to inactivate. Similar to DNA, RNA extraction involves using various buffers and enzymes to inactivate macromolecules and preserve the RNA.

Gel Electrophoresis

Because nucleic acids are negatively charged ions at neutral or basic pH in an aqueous environment, an electric field can mobilize them. Gel electrophoresis is a technique that scientists use to separate molecules on the basis of size, using this charge. One can separate the nucleic acids as whole chromosomes or fragments. The nucleic acids load into a slot near the semisolid, porous gel matrix’s negative electrode, and pulled toward the positive electrode at the gel’s opposite end. Smaller molecules move through the gel’s pores faster than larger molecules. This difference in the migration rate separates the fragments on the basis of size. There are molecular weight standard samples that researchers can run alongside the molecules to provide a size comparison. We can observe nucleic acids in a gel matrix using various fluorescent or colored dyes. Distinct nucleic acid fragments appear as bands at specific distances from the gel’s top (the negative electrode end) on the basis of their size (Figure 3). A mixture of genomic DNA fragments of varying sizes appear as a long smear; whereas, uncut genomic DNA is usually too large to run through the gel and forms a single large band at the gel’s top.

Nucleic Acid Fragment Amplification by Polymerase Chain Reaction

Although genomic DNA is visible to the naked eye when it is extracted in bulk, DNA analysis often requires focusing on one or more specific genome regions. Polymerase chain reaction (PCR) is a technique that scientists use to amplify specific DNA regions for further analysis (Figure 4). Researchers use PCR for many purposes in laboratories, such as cloning gene fragments to analyze genetic diseases, identifying contaminant foreign DNA in a sample, and amplifying DNA for sequencing. More practical applications include determining paternity and detecting genetic diseases.

DNA fragments can also be amplified from an RNA template in a process called reverse transcriptase PCR (RT-PCR). The first step is to recreate the original DNA template strand (called cDNA) by applying DNA nucleotides to the mRNA. This process is called reverse transcription. This requires the presence of an enzyme called reverse transcriptase. After the cDNA is made, regular PCR can be used to amplify it.

LINK TO LEARNING

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Deepen your understanding of the polymerase chain reaction by viewing this video.

Molecular Cloning

In general, the word “cloning” means the creation of a perfect replica; however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning.

Cloning small genome fragments allows researchers to manipulate and study specific genes (and their protein products), or noncoding regions in isolation. A plasmid, or vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA. In cloning, scientists can use the plasmid molecules to provide a “folder” in which to insert a desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, scientists call the DNA fragment from the human genome (or the genome of another studied organism) foreign DNA, or a transgene, to differentiate it from the bacterium’s DNA, or the host DNA.

Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). Scientists have repurposed and engineered plasmids as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. An important feature of plasmid vectors is the ease with which scientists can introduce a foreign DNA fragment via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that different commonly available restriction endonucleases can cut. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner. They are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two DNA strands, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, we call them “sticky ends.” Adding the enzyme DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, scientists can splice any DNA fragment generated by restriction endonuclease cleavage between the plasmid DNA’s two ends that has been cut with the same restriction endonuclease (Figure 5).

Recombinant DNA Molecules

Plasmids with foreign DNA inserted into them are called recombinant DNA molecules because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different molecule parts of the molecules can be traced back to different species of biological organisms or even to chemical synthesis. We call proteins that are expressed from recombinant DNA molecules recombinant proteins. Not all recombinant plasmids are capable of expressing genes. The recombinant DNA may need to move into a different vector (or host) that is better designed for gene expression. Scientists may also engineer plasmids to express proteins only when certain environmental factors stimulate them, so they can control the recombinant proteins’ expression.

VISUAL CONNECTION

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You are working in a molecular biology lab and, unbeknownst to you, your lab partner left the foreign genomic DNA that you are planning to clone on the lab bench overnight instead of storing it in the freezer. As a result, it was degraded by nucleases, but still used in the experiment. The plasmid, on the other hand, is fine. What results would you expect from your molecular cloning experiment?

1. There will be no colonies on the bacterial plate.
2. There will be blue colonies only.
3. There will be blue and white colonies.
4. There will be white colonies only.

^{Answer:
You’d expect the experiment would result in blue colonies only. (b.)}

LINK TO LEARNING

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View an animation of recombination in cloning from the DNA Learning Center.

CRISPR Technology

CRISPR technology has transformed the field of genetic engineering by providing a precise, efficient, and versatile method for editing genomes. At the heart of this innovation is the CRISPR-Cas9 system, a powerful tool derived from a natural defense mechanism found in bacteria (Figure 6).

CRISPR-Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas9 protein. This system uses a nuclease enzyme (Cas9) guided by RNA molecules to introduce breaks at specific DNA sequences.

The essential elements of this system are a guide RNA (gRNA) that homes in on the target sequence and a nuclease that can make a cut in the sequence that is bound by the guide RNA. By engineering guide RNAs complementary to a target gene, it is possible to target the nuclease to edit the gene in situ. In the CRISPR-Cas9 system, the Cas9 endonuclease cuts both strands of the gene sequence targeted by the guide RNA. This generates a double-strand break that the cell attempts to repair (Figure 7).

Double-strand breaks in DNA can be repaired by simple, nonhomologous end joining (NHEJ) or by homologous recombination. When a break is fixed by NHEJ, there is good chance that there will be deletions or insertions that will inactivate the gene they are in. Thus, targeted cleavage of a site by CRISPR-Cas9 can easily and specifically inactivate a gene, making it easy to characterize the gene’s function.

But, what if you wished to simply mutate the gene at a specific site to study the effect of the mutation? This, too, can be achieved. If a homologous sequence bearing the specific mutation (often referred to as a template strand or template gene) is provided homologous recombination can repair the break, and at the same time insert the exact mutation desired. It is obvious that if you can insert a mutation as just described, it should be possible to correct a mutation in the genome by cleaving at the appropriate spot and providing the correct sequence as a template for repair by homologous recombination. The simplicity of the system is what makes CRISPR-Cas9 such a powerful gene editing tool.

Biotechnology in Medicine and Agriculture

It is easy to see how biotechnology can be used for medicinal purposes. Knowledge of the genetic makeup of our species, the genetic basis of heritable diseases, and the invention of technology to manipulate and fix mutant genes provides methods to treat the disease. Biotechnology in agriculture can enhance resistance to disease, pest, and environmental stress, and improve both crop yield and quality.

Production of Vaccines, Antibiotics, and Hormones

Traditional vaccination strategies use weakened or inactive forms of microorganisms to mount the initial immune response. Modern techniques use the genes of microorganisms cloned into vectors to mass produce the desired antigen. Doctors then introduce the antigen into the body to stimulate the primary immune response and trigger immune memory. The medical field has used genes cloned from the influenza virus to combat the constantly changing strains of this virus.

Antibiotics are a biotechnological product. Microorganisms, such as fungi, naturally produce them to attain an advantage over bacterial populations. Cultivating and manipulating fungal cells produces antibodies.

Scientists used recombinant DNA technology to produce large-scale quantities of human insulin in E. coli as early as 1978. Previously, it was only possible to treat diabetes with pig insulin, which caused allergic reactions in humans because of differences in the gene product. In addition, doctors use human growth hormone (HGH) to treat growth disorders in children. Researchers cloned the HGH gene from a cDNA library and inserted it into E. coli cells by cloning it into a bacterial vector.

GMOs and Transgenic Organisms

Genetically Modified Organisms (GMOs) and transgenic organisms are vital in biotechnology for the production of complex proteins and for research purposes. While bacteria are commonly used to produce recombinant proteins, certain proteins or medical applications require modification of eukaryotic genomes. The term GMO refers to any organism, prokaryotic or eukaryotic, whose genome has been genetically altered. This could many having an existing gene deactivated or modified. Transgenic organisms are a type of GMO that have specifically had genetic material from another species edited into their genome, allowing them to express new traits or produce substances not naturally found in their species.

Manipulating the DNA of plants (i.e., creating GMOs) has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life (Figure 9). Plants are the most important source of food for the human population. Farmers developed ways to select for plant varieties with desirable traits long before modern-day biotechnology practices were established.

We call plants that have received recombinant DNA from other species transgenic plants. Because they are not natural, government agencies closely monitor transgenic plants and other GMOs to ensure that they are fit for human consumption and do not endanger other plant and animal life. Because foreign genes can spread to other species in the environment, extensive testing is required to ensure ecological stability. Staples like corn, potatoes, and tomatoes were the first crop plants that scientists genetically engineered.