PCR and Electrophoresis
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.
Polymerase Chain Reaction (PCR)
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.