Genetic Engineering Tools

Deoxyribonucleic acid (DNA) is microscopic, and cells contain only one or a few copies of each sequence. A single copy—or even several copies—of a piece of DNA is not enough to perform analyses on it. Often, it is necessary to amplify, or create many copies of, a piece of DNA. The polymerase chain reaction (PCR) is a technique for rapidly producing many copies of a section of DNA. A PCR reaction proceeds through several cycles where the target sequence is copied at each cycle, roughly doubling the number of sequences every cycle and rapidly creating many thousands to millions of copies. This exponential amplification quickly results in large quantities of the target sequence. For example, if there is one copy of the sequence at the start, after 20 cycles there are just over a million.

In a PCR reaction, the double stranded template DNA molecule, which is obtained from the larger DNA strand of the chromosome and contains the sequence for amplification, is mixed with several starting materials: primers, nucleotides, a replicating enzyme, and a buffer solution. Short single-stranded DNA sequences, called primers, that are complementary to areas flanking the target region of the template DNA are needed. Two primers are required: one complementing the 3′ end of one strand of the target region for amplification, and one complementing the 3′ end of the complementary strand on the opposite side of the target region. Everything between the locations of primer sequences will be copied. The nucleotide building blocks of DNA, deoxynucleoside triphosphate molecules, are required to build the new DNA molecules. DNA polymerase, the enzyme in DNA replication that assembles the new strands of DNA from the template strands, performs the reaction. A unique DNA polymerase from the bacterium Thermus aquaticus is utilized because it can withstand the high temperatures of PCR. All these molecules are mixed in a buffer solution containing stabilizing salts that provides an optimal environment for the DNA polymerase.

The reaction then proceeds through several steps that are cycled many times. In the first step, denaturation, the template DNA double helix heated to 95°C and is split into its two single-stranded components. In the annealing step, the temperature is lowered just enough to allow the primers to hybridize, or bind to complementary single stranded DNA sequence on the template strands. In the elongation step, the temperature is raised slightly where it is held long enough for the DNA polymerase, starting at the primers, to synthesize new strands complementary to the template. This completes one cycle. Amplification of a DNA sequence using PCR produces sufficient quantities of the molecules to conduct further analyses. Mixtures of DNA samples are separated by length using gel electrophoresis, a technique that separates DNA and other molecules based on their size using an electric charge. This technique takes advantage of the inherent negative charge on the phosphates of the sugar-phosphate backbone structure of DNA. The DNA is separated in a block of gelatinous material, typically made of agar and called a gel. The gel is placed in a conductive buffer solution and the DNA samples are loaded into cavities in the gel, called wells. An electrical field is created in the solution using a power source, and the DNA migrates toward the positive pole of the gel. Shorter DNA fragments travel farther through the gel since they move more easily through the pores in the material of the gel. After the differently sized fragments of DNA have been separated on the gel, they are stained with a chemical dye that binds to DNA and glows when excited with ultraviolet light. When visualized under ultraviolet light, distinct bands in the gel are revealed. Each band corresponds to a collection of DNA molecules with the same length. The separated DNA fragments are compared against a control or reference sample called a ladder composed of DNA fragments with known lengths. When the gel is visualized, the bands created by the ladder act like a ruler and are used to estimate the sequence lengths of the bands generated from samples.

DNA profiling is any of the techniques used for differentiating between two unrelated individuals' DNA. However, strands of DNA are very long. The genome of Escherichia coli consists of a single circular chromosome with 4,600,000 base pairs and 4,300 potential genes. Given this length, performing analyses on a whole chromosome is often impossible. Restriction fragment length polymorphism (RFLP) is a DNA profiling technique used to differentiate DNA sequences by cutting the sequences with restriction endonuclease enzymes and visualizing the varying lengths of the resultant restriction fragments. A restriction endonuclease is an enzyme that cuts DNA at specific locations. A small piece of DNA that results from cutting a larger piece of DNA with a restriction endonuclease is called a restriction fragment. Restriction endonucleases cut DNA by recognizing a target base pair sequence and cleaving the sugar-phosphate backbones of the DNA molecule at or adjacent to that target site. Thousands of restriction endonucleases have been discovered, each recognizing and cutting at a unique target sequence. In practice, restriction endonucleases may be used singly or in combinations to cut the DNA at multiple sites.

When RFLP is used for DNA profiling, the DNA samples being compared are cut with restriction endonucleases in separate reaction tubes. If the DNA samples are from different individuals they will have different sequences, yielding restriction fragments of differing lengths. The restriction fragments are separated by gel electrophoresis. Gel electrophoresis uses electricity to pull the DNA fragments through a gelatinous matrix of pores that act like a filter, and shorter DNA fragments travel farther into the gel, while longer gel fragments are retarded by the gel matrix and move slower. Researchers use a ladder as a control on the gel that serves as a ruler for how long the various DNA fragment bands may be on a gel.

The DNA fragments and the ladder in the gel are then transferred to a nylon film. The film is rinsed with a solution containing gene probes. A gene probe is a short sequence of DNA with a radioactive or fluorescent label. The gene probes hybridize, or bind with complementary single-stranded DNA sequences, on the film. Since the length of the gene probes is known, when they are visualized, the lengths of the restriction fragments are clear. When visualized, the resultant pattern of lengths from the sample creates a DNA profile. Patterns from different samples are compared to determine if they came from the same or different individuals. Samples from the same individual will have the same pattern. Restriction fragment length polymorphism is used to classify bacteria. Specifically, the gene that encodes the 16S ribosomal RNA gene is cut with restriction fragments. The 16S ribosomal RNA gene is present in all bacteria and its function has remained the same through evolutionary time. Differences in this highly conserved gene are suggested to be an accurate measure of evolutionary distance and are used for phylogenetic classification. Conserved sequences in other groups of organisms have similar applicability for deriving evolutionary relationships, for example chloroplast genes are used in plants and in fungi the 18S and 25S ribosomal RNA genes are used. In addition to classification, DNA profiling techniques can be used to conclusively identify a pathogen so that accurate treatment can be provided.