Biotechnology

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). 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.

This illustration shows the four main steps of DNA extraction. In the first step, cells in a test tube are lysed using a detergent that disrupts the plasma membrane. In the second step, cell contents are treated with protease to destroy protein, and RNAase to destroy RNA. The resulting slurry is centrifuged to pellet the cell debris. The supernatant, or liquid, containing the DNA is then transferred to a clean test tube. The DNA is precipitated with ethanol. It forms viscous, mucous-like strands that can be spooled on a glass rod
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.

Photo shows an agarose gel illuminated under UV light. The gel contains nine lanes from left to right. Each lane was loaded with a sample containing DNA fragments of differing size that separated as they travelled through the gel from top to bottom. The DNA appears as thin, white bands on a black background. Lanes one and nine contain many bands from a DNA standard. These bands are closely spaced toward the top, and spaced farther apart further down the gel. Lanes two through eight contain one or two bands each. Some of these bands are identical in size and run the same distance into the gel. Others run a slightly different distance, indicating a small difference in size. Image b shows a researcher working at a machine where she is observing DNA under ultraviolet light.
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.

Illustration shows the amplification of a DNA sequence by the polymerase chain reaction. PCR consists of three steps—denaturation, annealing, and DNA synthesis—that occur at high, low, and intermediate temperatures. In step 1, the denaturation step, the sample is heated to a high temperature so the DNA strands separate. In step 2, annealing, the sample is cooled so two primers can anneal to the two strands of DNA. The primers are spaced such that the sequence of interest between them will be amplified. In step 3, DNA synthesis, the sample is warmed to the optimal temperature for Taq polymerase, which synthesizes the complementary strand from the primer to the 3' end of the molecule. This cycle is repeated again and again. Each time, the newly synthesized strands serve as templates so that the amount of DNA doubles with each cycle. As the cycles continue, more and more strands are the size of the distance between the two primers; in the end, the vast majority of strands are this size.
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.

Link to Learning

Deepen your understanding of the polymerase chain reaction by clicking through this interactive exercise.

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.

In Southern blotting, DNA is separated on the basis of size by agarose gel electrophoresis. The fragments run through the gel from top to bottom. In the gel shown in this figure, there are so many DNA fragments they appear as a smear in each lane. The DNA from the gel is transferred to a nylon membrane. To do so, the gel is sandwiched between filter paper and the membrane and placed in hybridization buffer. Paper towels above the gel wick up the moisture and assist in the transfer. The nylon membrane is then incubated with a radioactively or fluorescently labeled probe that is complementary to the sequence of interest. Discrete bands appear where the sequence of interest is located.
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.
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