PCR Overview

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Polymerase Chain Reaction - PCR

PCR Components

PCR Process

Stages of PCR

Visualization of the Amplicon

Reagents specific to RT-PCR

One-step and Two-step RT-PCR

Quantitative PCR

Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR)

Hot Start PCR

High Fidelity PCR

References

 

Polymerase Chain Reaction - PCR

Polymerase chain reaction (PCR) is a technique for detecting, quantifying and amplifying nucleic acids. The basic PCR mechanism involves the use of an enzyme called DNA polymerase to synthesize complementary strands of DNA from a denatured double-stranded template, effectively doubling the original sample with every cycle of the PCR reaction. This molecular copying operates through cycles of thermal reactions enabled by an assembly of biochemical reagents, and amplifies a few copies of DNA to millions. Amplified DNA can then be analyzed qualitatively (for its presence), quantitatively (by amount) or sequentially (for its genetic code), and used in downstream molecular biology applications.

Since its development by Kary Mullis in 1983, PCR has revolutionized the molecular biology field, giving rise to many advantageous techniques that allow the analysis of different nucleic acids.

 

PCR Components

It’s important to be familiar with the materials and reagents involved in PCR and understand how altering their concentration in the PCR reaction can lead to optimal target DNA amplification.

• Template: This is the sample of nucleic acids that contain the desired target sequence, and is separated initially into two strands in PCR reactions. The sample can originate from different sources including (but not limited to) human blood, saliva, skin or hair and cells from other smaller organisms, such as microbes and its purity must be guaranteed for accurate qualitative analysis. Optimal amounts of template will depend on composition and source of the deoxyribose nucleic acid (DNA) template and the type of polymerase used. Generally, PCR requires 1-1000 ng (104-107 molecules) of DNA template.
• For viral and plasmid templates, 1-1000 pg template is sufficient.
• For genomic DNA and more complex templates, 1-1000 ng is recommended.
• Usually, for a 50 µl cloning reaction, 100-250 ng of mammalian genomic DNA or 20 ng of linearized plasmid DNA is recommended.

• Primer pairs: Primers are short oligonucleotides (synthetic short DNA strand), which complement the target sequence and bind to the single-stranded DNA. These primers are short compared to the template, only 20-40 nucleotides long (18 nucleotides minimum). Pairs of primers are employed in each reaction, but they should have melting temperature (Tm) values within 5°C of each other. Large variation in the Tm of a primer pair results in poor amplification. Primers should contain 50% GC (guanosine-cytosine content)-content (Tm 56-62°C), ideally. This content predicts their annealing temperature to the template; higher GC-levels correlate with a high Tm. A lower Tm will result in nonspecific products and a high Tm will result in decreased amplicon yield. In addition, perfect base pairing between the template and the 3' end of the primer is critical. Each reaction should contain no more than 0.05-0.1µM of each primer at final concentration.

• dNTPs: DNA polymerase requires deoxynucleotide triphosphates – also known as dNTPs – the nucleic acid components, or nucleotide bases - adenine, thymine, cytosine and guanine (A, T, C, G) - to generate new DNA. In general, 20-200μM (50μM of each is ideal) of each dNTP should be included in the reaction. Excess dNTPs inhibits polymerase activity; whereas low dNTP concentration enhances the fidelity of polymerization but reduces the amplicon yield. Longer target sequences may require an increase in dNTP concentration. However, one must keep in mind that as dNTP concentration increases, so does the amount of (Mg²⁺) needed for optimal amplification.

• DNA polymerase: DNA polymerase is a thermostable enzyme that synthesizes copies of DNA with every consecutive cycle of PCR. Polymerases are responsible for binding free nucleotides complementary to the template DNA. The enzyme first binds and adds a nucleotide to the 3'-OH group of the primer. The polymerase then moves in a 5'->3' direction, constantly adding dNTPs. It’s necessary for the polymerase to come from alternate organisms from humans or other common vertebrates so the enzyme won’t be broken down at the temperatures required to denature DNA during the PCR reaction. As such, bacteria from hot springs have provided some of these polymerases, including Taq and Pfu. In an average amplifying reaction, 0.5-2.0 units of polymerase should be used for every 50 µl reaction mixture. Ideally, 1.25 units total is recommended.

• Cofactors and buffer: Magnesium (Mg²⁺) is an important cofactor for DNA polymerase. Magnesium assists phosphodiester bond formation and is required for successful PCR amplification. Mg²⁺ concentration should be maintained at around 0.5-5.0mM in reactions using Taq polymerase. Usually, the concentration is higher than that of dNTPs and primers, and must be optimized and determined by the template, buffer and dNTP content. In addition, some polymerases may require as high as 6mM Mg²⁺ while others need only ≤ 1mM Mg²⁺. Magnesium concentration can be changed to optimize PCR amplification. Increasing magnesium concentration results in higher product yield. However, an increase in this cofactor also results in a decreased DNA polymerase specificity and fidelity. Potassium salt (K+), present in the buffer, can also be modified to improve PCR amplification. Usually, in the buffer should be 35-100mM. It's important to note that amplification of a long PCR product may require a decrease in K+. The buffer in a PCR reaction serves to facilitate amplification by stabilizing the polymerase. Different PCR buffers are currently used, but the preferred one is Tris/HCl, which must maintain a pH of 8.4 at room temperature.

The PCR reaction may be enhanced through the use of additives. These may increase stringency or stabilize enzymatic activity resulting in increase PCR efficiency. Various additives along with their effects and recommended concentration are discussed in the following table.

PCR Process

Basic PCR can be split into three general stages: denaturation, annealing and extension. Typically, a PCR protocol consists of an initial denaturation step, around 30 cycles of these three stages, a final extension step, and a holding step with a temperature of 4-10°C. Shown below is one PCR cycle detailing the different stages and the corresponding temperatures.

Stages of PCR

• Denaturation: In the first stage, the denaturation stage, double-stranded DNA is separated into two single strands by breaking the hydrogen bonds between the nucleotide base pairs (bp). In a PCR reaction, an initial denaturation step is needed at the beginning of PCR, before the cycling of the three stages begins. This initial denaturation step usually occurs at around 94°C (or in the range of 94-98°C), depending on the specific polymerase involved. To fully denature the DNA, initial denaturation can take up to 2 minutes. A higher GC content requires higher and longer denaturation temperatures. However, it’s better to avoid extended incubations at this denaturation temperature to prevent inactivation of the polymerase or damage to the DNA template. Once this initial denaturation step is completed, thermal cycling of denaturation, annealing and extension can begin. The denaturation step is usually set at the same temperature as initial denaturation, ~94°C, and will require only 10-60 seconds.

 

• Annealing: Annealing is the next stage of the PCR process. In this step, primers anneal, or bind, at complementary sequences of the denatured DNA strands. As previously stated, this will only occur successfully if temperature and primer pairs have been optimized. The annealing temperature (Ta) should be 5°C below the lowest Tm of either primer. Ideally, Ta falls between 52-58°C, and the annealing time is maintained for 15-60 seconds, with optimum time at 30 seconds. If the annealing temperature is too high, primers do not anneal efficiently; if it’s too low, primers may bind nonspecifically to the template.
  

 

• Extension: Extension is the final stage of a cycle and occurs at a temperature that allows optimal polymerase activity of binding nucleotides to the annealed primer resulting in exponential amplification of the template strand. Usually, the extension step can be carried out at 70-80°C for 1 to 2 minutes. Extension temperature and time are specific to the polymerase. For example, Taq operates best at 70-80°C for 1 minute for the first 2 kb, and requires another minute per additional kb; Pfu, meanwhile, functions at 75°C and requires 2 minutes per 1kb of DNA. Overextending this phase will create higher error rates. Annealing and extension can be compressed into one stage if the corresponding temperatures are similar, and this is referred to as “two-step” PCR, because the three stages have been reduced to two. Two-step PCR is most often used when the Tm of primers are within the normal range for extension activity. One final extension period of 5 minutes is performed at the end of thermal cycling, which results in addition of adenines to the 3' ends of amplicons. This residue is needed for different downstream applications including cloning.

 

Visualization of the Amplicon

The resulting amplicon can then be visualized through staining with dye or through labeling with fluorescent nucleotides or primers. In an electrophoresis reaction, agarose gels and visualizing molecules demonstrate the size and quantity of DNA product in comparison to DNA ladders (molecular weight size markers) with standard lengths and markers.

This visualization is conducted in reactions like gel electrophoresis, a standard procedure that separates DNA molecules via length and demonstrates their homogeneity. Electrical fields cause DNA to migrate through a gel matrix with differential speeds, and they can be tagged with other molecules for easy identification through color or fluorescence. Shorter fragments move at a more rapid pace, so approximate size can be calculated by the comparison between PCR fragments and DNA ladders to confirm consistency.

Various specialized PCR techniques have been developed to address researchers’ specific needs. The use of a specific type of PCR depends on the nucleic acid source, the desired amplicon and downstream application. These distinct techniques may involve a supplementary reaction to amplify target sequences from RNA. Others employ a different mechanism of analysis or amplicon visualization, such as when the product is quantified during the replication cycles. Here, we discuss popular variations on this method including reverse transcriptase PCR (RT-PCR), quantitative PCR (qPCR) and reverse transcriptase quantitative PCR (RT-qPCR).

 

Reverse transcription PCR, or RT-PCR, is used to create an amplicon from a sample of RNA rather than DNA. In this technique, RNA is first reverse transcribed into double-stranded complementary DNA (cDNA), which is then amplified through PCR. As with basic PCR, the purity and integrity of the starting sample is critical in RT-PCR because successful amplification relies on proper reverse transcription of RNA into cDNA. Furthermore, the additional steps in RT-PCR increases the potential for contamination, degradation or mistakes to proliferate and must be considered

 

Reagents specific to RT-PCR

• Reverse Transcriptase: This enzyme contains DNA polymerase activity and is routinely used to generate cDNA from RNA. This enzyme may also contain ribonuclease H (RNaseH) activity, which allows for the cleaving and degradation of the RNA strand in the DNA/RNA hybrid after transcription is completed. Reverse transcriptases in RT-PCR are thermostable and able to generate cDNA with high processivity after the primer anneals to the RNA template. The most commonly used reverse transcriptases are Moloney Murine leukemia virus reverse transcriptase (M-MLV RT) and avian myeloblastosis virus enzyme (AMV RT). AMV RT is more thermally stable than M-MLV allowing for its use at higher temperatures.

• RT-PCR Primers:
• Sequence-specific primers attach only to the region of the template’s genetic code complementary to their own and begin replicating in the 5’-end direction. These primers are commonly used in one-step RT-PCR.
• Oligo(dT) primers are usually 12-16 bp long and contain repeated deoxythymines in their 3’ end that bind to the poly(A) tail at the 3’-end of the DNA strand. These primers proceed to amplify in the 5' end direction similarly to gene-specific primers. When a long amplicon or full-length transcript is desired, either gene-specific or oligo(dT) primers are favored. Oligo(dTs) cannot be used to transcribe RNA lacking poly (A) tails, degraded RNA or RNA that has considerable secondary structures.
• Random primers bind themselves less specifically to the template with multiple primers attached to one strand, and amplification progresses towards the 5’-end from each primer, terminating whenever the point of replication from another primer is reached. Random primers work well on degraded samples of RNA. To excise and replicate just a portion of a longer transcript, random oligomer primers or oligo(dT) with random nonamers will produce cDNA for portions of a full-length transcription.

• Magnesium (Mg²⁺) and manganese (Mn²⁺): Divalent cations, magnesium and manganese, both serve as cofactors in a reverse transcription reaction. Magnesium may be replaced with manganese to properly activate specific polymerases including AMV DNA polymerase. It should be noted that excess manganese will have the same decrease specificity as excess magnesium does.

 

One-step and Two-step RT-PCR

RT-PCR can be performed using a one-step or a two-step method. In one-step RT-PCR the reagents for the RT and PCR reaction are added to a single tube allowing both reactions to occur simultaneously. In two-step RT-PCR, the RT reaction occurs separately from the PCR reaction. One advantage of one step RT-PCR is the lower probability of contamination because the sample is less exposed to the environment in a single tube and the process requires less pipetting. Also, the results have higher reproducibility and the procedure takes less time. However, one-step RT-PCR only allows the use of sequence-specific primers. On the other hand, two-step RT-PCR allows the use of random primers or oligo(dTs) and the resulting cDNA sample can be stored for long periods of time and used for multiple PCRs. The cDNA template, dNTPs, polymerases and salts are transferred from the finished RT reaction to the amplifying reaction (see One-step and two-step RT-PCR Figure). This increases the chances for contamination (further discussed in the Troubleshooting section).

 

Quantitative PCR

PCR is a very useful method for qualitative DNA analysis and for the amplification of less abundant DNA samples for sequencing, cloning, genotyping and other applications. In another PCR method, quantitative PCR (qPCR), also known as real time quantitative PCR (RT-qPCR) and quantitative real time PCR (qRT-PCR), we can analyze the quantity (copy number) of target DNA as it is replicated, in real time. PCR amplification occurs in different phases that are evident in the resulting qPCR amplification curve (shown in green): the initiation phase, exponential growth phase and the plateau.

Quantification of the amplicon is possible due to the use of fluorescent emission from a dye or probe upon production of double stranded DNA. When amplification is just beginning, at the initiation phase, very low levels of fluorescence (baseline levels) from the initial reporter are detected because a very small amount of amplicon has been produced. The reaction is just beginning and any small levels of fluorescence generated are considered noise at this stage. A threshold value, the point at which fluorescence reaches values above baseline levels can be set manually or by the analysis software being used. In the following stage, the exponential growth phase, the subsequent successive cycles of amplification results in exponential increase of copies of the target DNA, leading to accumulation of fluorescence. During this stage the quantity of the product is directly proportional to amount of template and it's when quantification analysis is performed. In the third phase, the plateau, amplification is no longer occurring exponentially since reagents have been used and fluorescence levels do not increase. This method is routinely used to specifically quantify the amount of one target gene, fractions of DNA or other organic molecules within one sample.

qPCR uses a fluorescent reporter molecule – a fluorescent dye, labeled oligonucleotide primer or probe.

• A dye binds double stranded DNA nonspecifically. The most commonly used one is SYBR Green. Melt curve analysis is performed at the end of reaction involving a dye, allowing the assessment of the quality and characteristics of the resulting amplicon. Optimally, only the targeted region of a template is amplified during qPCR, resulting in a specific product. At the end of the reaction, the dye is bound to dsDNA (the amplicon) and fluorescence levels are high. To generate the melting curve, the temperature is lowered causing dissociation of ds DNA and a decrease in fluorescence. The resulting melting peak can then be analyzed.

 

• If a primer or probe is employed, two reporters are involved, and a proximity-based energy transfer is used to identify the desired PCR product. Energy transfer techniques are a sensitive mode of detection. The first reporter’s energy is absorbed in the reaction and causes the second reporter to respond in such a way that it confirms DNA accumulation through heat emission or fluorescent resonance energy transfer (depending on the identity of the second reporter). Essentially, light moves from the first reporter to the second reporter. Probes come in specific forms for complementing nucleic acids on the target sequence. The available range of complexity and identity of these reporters is extensive, but all probes are capable of annealing to amplicons at specific target regions.
  

 

Reverse Transcriptase quantitative Polymerase Chain Reaction (RT-qPCR)

Quantification of a specific RNA target can be achieved by coupling reverse transcription (RT) with qPCR – reverse transcriptase real time quantitative PCR. This method requires the collection of RNA, then the reverse transcription to obtain cDNA followed by quantification with real-time quantitative PCR. The polymerase must be carefully chosen and primers must be designed for proper reverse transcription of target RNA. In addition, the resulting cDNA must correspond to the entire RNA template provided for accurate quantification. Other specialized protocols and kits are available for mRNA or microRNA RT-qPCR.

RT-qPCR can be performed in either one- or two-step procedures described previously. Thus, the summarized steps of RT-qPCR are

1. Generation of cDNA through reverse transcription.

2. Quantitative PCR and thermocycling.

3. Data output for analysis.

 

Hot Start PCR

Hot start method of PCR is a technique that is often used to improve PCR efficiency by preventing nonspecific product formation. This technique involves excluding one or more reagents (such as the polymerase) until a specific temperature (60-80°C) is reached or reversibly inhibiting the polymerase. In the first method, as the reaction is assembled at specific temperature, the missing reagent is added after a specific temperature is reached. In another hot start method, when all the materials are combined, the temperature is increased to a heat index that will prompt polymerase activity, which was previously inactive due to its bonds to another molecule. Another approach involves chemically modifying the primers may be modified. Also other reagents might be retained in wax, so that heat is required to free them for use in the reaction.

Hot start prevents polymerases like Pfu or Taq from extending before a higher temperature is reached, thus decreasing the likelihood of nonspecific transcription at low temperatures. The method benefits small and large-quantity replication by stalling polymerase activation until covalent bonds are broken during denaturing. Hot start PCR will improve both multiplex and single-sided PCR reactions by suppressing these side reactions, primer oligomerization and mis-priming that reduce amplicon production. Less primer dimerization or nonspecific DNA annealing improves PCR product; starting when high heat is already achieved also prevents side products from being rapidly produced as they are at room temperature, as these can impede amplicon generation.

 

High Fidelity PCR

High fidelity PCR is useful when the downstream applications include cloning, mutagenesis and expression, which require minimal errors during the replication process. Pfu is a commonly used proofreading polymerase used in high fidelity PCR. This enzyme has 3′→5′ exonuclease activity and is used to reduce the number of replication errors. Pfu's exonuclease activity corrects mismatches between the template and the new DNA strand by cleaving the erroneous nucleotide and replacing it with the correct one. The error rate of Pfu is 10-fold lower than that of Taq polymerase, and it is capable of higher fidelity at high pH. In addition, Pfu can amplify up to 25 kb. Proofreading activity does slow down the process, but it allows the removal of an incorrect nucleotide.

Multiplex PCR is another common variant which uses differing pairs of primers for the same reaction in multiple iterations, a technique that amplifies different target sequences from within the same sample. In this method, optimization is of larger concern to effectively amplify the various targets, so hot-start method and optimizing buffers often coincide with multiplex techniques. The process requires labeled oligonucleotide primers or probes when employed for qPCR. Due to the complexity of the multiple-tiered transcription, materials for multiplex are often assembled in kits for specifically designed tests and can be purchased as such.

There are numerous other variations on PCR, including nested, long-range and single-cell, but they are less frequently used. Much of the protocol for the more common systems can be found on GoldBio’s website, and we list them below for easy access.

 

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