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)). 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.

Figure 2. This diagram shows the basic method of DNA extraction.

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)). 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.

Figure 3. a) Shown are DNA fragments from seven samples run on a gel, stained with a fluorescent dye, and viewed under UV light; and b) a researcher from International Rice Research Institute, reviewing DNA profiles using UV light. (credit: a: James Jacob, Tompkins Cortland Community College b: International Rice Research Institute)

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)). 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.

Figure 4. Scientists use polymerase chain reaction, or PCR, to amplify a specific DNA sequence. Primers—short pieces of DNA complementary to each end of the target sequence combine with genomic DNA, Taq polymerase, and deoxynucleotides. Taq polymerase is a DNA polymerase isolated from the thermostable bacterium Thermus aquaticus that is able to withstand the high temperatures that scientists use in PCR. Thermus aquaticus grows in the Lower Geyser Basin of Yellowstone National Park. Reverse transcriptase PCR (RT-PCR) is similar to PCR, but cDNA is made from an RNA template before PCR begins.

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.

Hybridization, Southern Blotting, and Northern Blotting

Scientists can probe nucleic acid samples, such as fragmented genomic DNA and RNA extracts, for the presence of certain sequences. Scientists design and label short DNA fragments, or probes with radioactive or fluorescent dyes to aid detection. Gel electrophoresis separates the nucleic acid fragments according to their size. Scientists then transfer the fragments in the gel onto a nylon membrane in a procedure we call blotting ((Figure)). Scientists can then probe the nucleic acid fragments that are bound to the membrane’s surface with specific radioactively or fluorescently labeled probe sequences. When scientists transfer DNA to a nylon membrane, they refer to the technique as Southern blotting. When they transfer the RNA to a nylon membrane, they call it Northern blotting. Scientists use Southern blots to detect the presence of certain DNA sequences in a given genome, and Northern blots to detect gene expression.

Figure 5. Scientists use Southern blotting to find a particular sequence in a DNA sample. Scientists separate DNA fragments on a gel, transfer them to a nylon membrane, and incubate them with a DNA probe complementary to the sequence of interest. Northern blotting is similar to Southern blotting, but scientists run RNA on the gel instead of DNA. In Western blotting, scientists run proteins on a gel and detect them using antibodies.

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.

Art Connection

Figure 6. This diagram shows the steps involved in molecular cloning.

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. The will be white colonies only.

Gene Targeting

Although classical methods of studying gene function began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: “What does this gene or DNA element do?” This technique, reverse genetics, has resulted in reversing the classic genetic methodology. This method would be similar to damaging a body part to determine its function. An insect that loses a wing cannot fly, which means that the wing’s function is flight. The classical genetic method would compare insects that cannot fly with insects that can fly, and observe that the non-flying insects have lost wings. Similarly, mutating or deleting genes provides researchers with clues about gene function. We collectively call the methods they use to disable gene function gene targeting. Gene targeting is the use of recombinant DNA vectors to alter a particular gene’s expression, either by introducing mutations in a gene, or by eliminating a certain gene’s expression by deleting a part or all of the gene sequence from the organism’s genome.

Genetic Diagnosis and Gene Therapy

Scientists call the process of testing for suspected genetic defects before administering treatment genetic diagnosis by genetic testing. Depending on the inheritance patterns of a disease-causing gene, family members are advised to undergo genetic testing. For example, doctors usually advise women diagnosed with breast cancer to have a biopsy so that the medical team can determine the genetic basis of cancer development. Doctors base treatment plans on genetic test findings that determine the type of cancer. If inherited gene mutations cause the cancer, doctors also advise other female relatives to undergo genetic testing and periodic screening for breast cancer. Doctors also offer genetic testing for fetuses (or embryos with in vitro fertilization) to determine the presence or absence of disease-causing genes in families with specific debilitating diseases.

Gene therapy is a genetic engineering technique used to cure disease. In its simplest form, it involves the introduction of a good gene at a random location in the genome to aid the cure of a disease that is caused by a mutated gene. The good gene is usually introduced into diseased cells as part of a vector transmitted by a virus that can infect the host cell and deliver the foreign DNA ((Figure)). More advanced forms of gene therapy try to correct the mutation at the original site in the genome, such as is the case with treatment of severe combined immunodeficiency (SCID).

Figure 8. Gene therapy using an adenovirus vector can be used to cure certain genetic diseases in which a person has a defective gene. (credit: NIH)

Transformation of Plants Using Agrobacterium tumefaciens

Gene transfer occurs naturally between species in microbial populations. Many viruses that cause human diseases, such as cancer, act by incorporating their DNA into the human genome. In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by DNA transfer from the bacterium to the plant. Although the tumors do not kill the plants, they stunt the plants and they become more susceptible to harsh environmental conditions. A. tumefaciens affects many plants such as walnuts, grapes, nut trees, and beets. Artificially introducing DNA into plant cells is more challenging than in animal cells because of the thick plant cell wall.

Researchers used the natural transfer of DNA from Agrobacterium to a plant host to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids, Ti plasmids (tumor-inducing plasmids), that contain genes to produce tumors in plants. DNA from the Ti plasmid integrates into the infected plant cell’s genome. Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome. The Ti plasmids carry antibiotic resistance genes to aid selection and researchers can propagate them in E. coli cells as well.

The Organic Insecticide Bacillus thuringiensis

Bacillus thuringiensis (Bt) is a bacterium that produces protein crystals during sporulation that are toxic to many insect species that affect plants. Insects need to ingest Bt toxin in order to activate the toxin. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin activates in the insects’ intestines, they die within a couple of days. Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects. Scientists have cloned the crystal toxin genes from Bt and introduced them into plants. Bt toxin is safe for the environment, nontoxic to humans and other mammals, and organic farmers have approved it as a natural insecticide.

Flavr Savr Tomato

The first GM crop on the market was the Flavr Savr Tomato in 1994. Scientists used antisense RNA technology to slow the softening and rotting process caused by fungal infections, which led to increased shelf life of the GM tomatoes. Additional genetic modification improved the tomato’s flavor. The Flavr Savr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.