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When I was a graduate student, we called the lab’s PCR machine the “Magic Box,” because the reagents that we put inside could produce exactly what we were looking for, something completely unexpected, or nothing at all. If a reaction did not work, it was often difficult to tell exactly what needed to be changed. Was it the primers? The template? The shoes you were wearing that day?
Understanding what is happening in that Magic Box can ensure that you are able to design successful PCR experiments, and this article will dive into some of the tips we have learned at VectorBuilder.
What happens when you press “Start”
Polymerase chain reaction (PCR) is the gold standard in molecular biology labs, because it allows scientists to produce a huge number of copies of DNA fragments in a short time. Having many copies of this piece of DNA is important for efficient ligation of the fragment into a plasmid, so that the DNA can be introduced into cells or animals. PCR has countless other downstream applications, including genotyping, quantification of gene expression, and detection of pathogens like COVID19.
In order to amplify a specific sequence of DNA, the reaction requires a template to be amplified, primers that are complementary to the 5’ and 3’ ends that surround the target sequence, nucleotides, salts (including Mg2+), and a DNA polymerase. If all goes well, once that tube enters the machine, magic happens:
Figure 1. Steps in polymerase chain reaction (PCR)
The target DNA is denatured at about 95°C which causes the hydrogen bonds that hold the complementary strands of DNA together to break. The temperature is then dropped to the annealing temperature, typically about 50-60°C, at which primers bind to the complementary DNA sequence. Finally, the DNA polymerase forms a new complementary strand through extension, adding complementary nucleotides one at a time. After the first cycle, the newly made complementary strand can serve as an additional template to which primers bind, allowing exponential amplification (Figure 1). After about an hour, the PCR tube looks the same but the organization of the world inside is vastly different (assuming you were wearing the right shoes).
Your plan after the reaction is complete is an extremely important factor in deciding what reagents and type of PCR to use. Are you examining gene expression? Determining if a gene or pathogen is present in your sample? Introducing mutations to study gene function? Performing next generation sequencing?
The wide world of PCR templates and applications
One of the most straight-forward examples of PCR uses a DNA template (plasmid or genomic DNA) to amplify a fragment for either detection or cloning. This can be used to determine if a certain allele or gene is present in your genomic sample or to amplify a fragment for cloning into a plasmid. Additionally, a plasmid template can be used for downstream processes like mutagenesis or in situ hybridization probe synthesis, which allows visualization of gene expression in cells and tissues. Specialized primers can be used in these experiments in preparation for next generation sequencing (NGS).
A genomic DNA template can also be used in quantification experiments through quantitative PCR (qPCR, or real-time PCR). This allows you to determine the amount or copy number of a particular template sequence, for instance when detecting E. coli contamination. In qPCR, probes fluoresce as double-stranded DNA is formed, resulting in live tracking of amplification and quantification of the original sequence.
When RNA is used as a template, reverse transcription (RT) must occur as part of the PCR process, giving us RT-PCR. This alone has relatively limited applications: primarily preparation of a cDNA library or cloning of a coding sequence. Processed total RNA or mRNA for a gene of interest is reverse transcribed into cDNA fragments, which can then be sequenced/screened or cloned into a plasmid, respectively.
Often when using RNA as a template, reverse transcription is the first step in quantitative PCR. RT-qPCR allows for quantification of gene expression, an important aspect of experiments including RNAi validation, viral titer determination, and detection of diseases ranging from COVID-19 to cancer.
The following table highlights some of the differences in various PCR approaches.
| PCR | qPCR | RT-PCR | RT-qPCR | |
|---|---|---|---|---|
| Typical template | DNA (plasmid or genomic) | DNA (genomic) | RNA | RNA |
| Product | DNA | DNA | cDNA | cDNA |
| Quantifiable | No | Yes | No | Yes |
| Application | Detection of gene/allele; cloning | Detection of level of DNA pathogens; detection of copy number of genes | cDNA library synthesis; cloning of coding sequence | Detection of level of gene expression |
When PCR goes wrong
Each of the reagents that go into a PCR reaction must be optimized theoretically and/or empirically. When the reaction yields nothing or, often worse, unexpected bands, it can be difficult to determine which reagent(s) or setting must change.
Typically, the first variables to target are the primers and their annealing temperatures. It is crucial to ensure that primers are designed with high specificity to the target sequence, with appropriate GC content (40-55%), lacking repetitive sequences, and with no formation of secondary structures. Although primers have a theoretical annealing temperature, it may need to be optimized empirically, by running reactions with varying annealing temperatures and analyzing output on a gel. Additionally, if extra nucleotides are added to the primer to facilitate downstream experiments like sequencing, primer and annealing conditions often need to be optimized as well.
Templates must also be considered when designing PCR experiments. For example, plasmids should better be linearized because DNA is typically more accessible by both primers and the polymerase compared to circular DNA. Additionally, concentration and purity must be optimized.
The table below lays out guidelines for various components of your PCR reaction.
| Primers | Template | Mg2+ | |
|---|---|---|---|
| Design/Input | Check compatibility with 2+ DNA polymerase for source (MgCl2 vs MgSO4) | ||
| PCR Setup | Consider reaction components (EDTA and increased dNTPs require higher Mg2+) | ||
| Concentration | 0.05-1 uM | Plasmids: 0.1-1 ng Genomic/library DNA: 5-50 ng | 1-4 mM |
If your PCR is not working, producing either weak or nonspecific bands, and your primers and annealing temperatures have been optimized, it can be tempting to just increase everything: your template, your primers, the number of cycles. However, often less is more. Especially when dealing with complex templates, such as library DNA, the interactions of the hundreds and thousands of different molecules become highly unpredictable, and having too many cycles or too much template can result in production of artifacts and inefficient amplification of your target products. Eventually, the reaction passes the exponential phase of amplification, primers are exhausted, and the denatured DNA can form heteroduplex structures, which can appear as weak or nonspecific bands. Nonspecific bands can also form because of partial-length products. These shorter single-stranded products can bind to any complementary single-stranded DNA and can even act as primers themselves, forming undesirable products.
While the Magic Box can bring heartache, it is undoubtably full of wonder. Countless innovations, medicines, and treatments would not be possible without PCR. Yes, this seemingly basic procedure can present challenges, but VectorBuilder’s vast experience can take care of your cloning and library projects for you.
Sources
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