Abstract
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery. For example, the field of microbiology was transformed with the advent of Anton van Leeuwenhoek's microscope, which allowed scientists to visualize prokaryotes for the first time. The development of the polymerase chain reaction (PCR) is one of those innovations that changed the course of molecular science with its impact spanning countless subdisciplines in biology. The theoretical process was outlined by Keppe and coworkers in 1971; however, it was another 14 years until the complete PCR procedure was described and experimentally applied by Kary Mullis while at Cetus Corporation in 1985. Automation and refinement of this technique progressed with the introduction of a thermal stable DNA polymerase from the bacterium Thermus aquaticus, consequently the name Taq DNA polymerase.PCR is a powerful amplification technique that can generate an ample supply of a specific segment of DNA (i.e., an amplicon) from only a small amount of starting material (i.e., DNA template or target sequence). While straightforward and generally trouble-free, there are pitfalls that complicate the reaction producing spurious results. When PCR fails it can lead to many non-specific DNA products of varying sizes that appear as a ladder or smear of bands on agarose gels. Sometimes no products form at all. Another potential problem occurs when mutations are unintentionally introduced in the amplicons, resulting in a heterogeneous population of PCR products. PCR failures can become frustrating unless patience and careful troubleshooting are employed to sort out and solve the problem(s). This protocol outlines the basic principles of PCR, provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction. By following this PCR guide, students should be able to: ● Set up reactions and thermal cycling conditions for a conventional PCR experiment ● Understand the function of various reaction components and their overall effect on a PCR experiment ● Design and optimize a PCR experiment for any DNA template ● Troubleshoot failed PCR experiments
Highlights
In the biological sciences there have been technological advances that catapult the discipline into golden ages of discovery
This protocol outlines the basic principles of polymerase chain reaction (PCR), provides a methodology that will result in amplification of most target sequences, and presents strategies for optimizing a reaction
There are a few common problems that arise when designing primers: 1) self-annealing of primers resulting in formation of secondary structures such as hairpin loops (Figure 1a); 2) primer annealing to each other, rather the DNA template, creating primer dimers (Figure 1b); 3) drastically different melting temperatures (Tm) for each primer, making it difficult to select an annealing temperature that will allow both primers to efficiently bind to their target sequence during themal cycling (Figure 1c) (See the sections CALCULATING MELTING TEMPERATURE (Tm) and MODIFICATIONS TO CYCLING CONDITIONS for more information on Tms)
Summary
Designing appropriate primers is essential to the successful outcome of a PCR experiment. 4. The 3' ends of a primer set, which includes a plus strand primer and a minus strand primer, should not be complementary to each other, nor can the 3' end of a single primer be complementary to other sequences in the primer. The 3' ends of a primer set, which includes a plus strand primer and a minus strand primer, should not be complementary to each other, nor can the 3' end of a single primer be complementary to other sequences in the primer These two scenarios result in formation of primer dimers and hairpin loop structures, respectively. 6. Di-nucleotide repeats (e.g., GCGCGCGCGC or ATATATATAT) or single base runs (e.g., AAAAA or CCCCC) should be avoided as they can cause slipping along the primed segment of DNA and or hairpin loop structures to form. In order to avoid amplification of related pseudogenes or homologs it could be useful to run a blast on NCBI to check for the target specificity of the primers
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