The difference between PCR, RT-PCR and qPCR

As PCR is a highly sensitive method and very small volumes are required for single reactions, the preparation of a master mix for several reactions is recommended. The master mix must be well mixed and then split by the number of reactions, ensuring that each reaction will contain the same amount of enzyme, dNTPs, and primers. Our supplier Enzo Life Sciences offers PCR mixes that already contain everything except primers and the DNA template.

Guanine/Cytosine-rich (GC-rich) regions represent a challenge in standard PCR techniques. GC-rich sequences are more stable than sequences with lower GC content. Furthermore, GC-rich sequences tend to form secondary structures, such as hairpin loops. As a result, GC-rich double strands are difficult to completely separate during the denaturation phase. Consequently, DNA polymerase cannot synthesize the new strand without hindrance. A higher denaturation temperature can improve this, and adjustments towards a higher annealing temperature and shorter annealing time can prevent the unspecific binding of GC-rich primers. Additional reagents can enhance the amplification of GC-rich sequences. DMSO, glycerol, and betaine help to disrupt the secondary structures that are caused by GC interactions and thereby facilitate the separation of the double strands.

Unspecific amplification is a problem that can occur during PCR. Most DNA polymerases that are used for PCR work best at temperatures around 68°C to 72°C. The enzyme can, however, also be active at lower temperatures, although to a lower degree. At temperatures far below the annealing temperature, primers can bind non-specifically and lead to non-specific amplification, even if the reaction is set up on ice. This can be prevented by using polymerase inhibitors that dissociate from the DNA polymerase only once a certain temperature is reached, hence the term hot start PCR. The inhibitor can be an antibody that binds the polymerase and denatures at the initial denaturation temperature (95°C typically).

While DNA polymerases amplify fairly accurately to the original template sequence, mistakes in nucleotide matching can occur. Mismatches in applications such as cloning can result in truncated transcripts, and mistranslated or inactive proteins downstream. To avoid these mismatches, polymerases with a “proofreading” activity have been identified and incorporated into the workflow. The first proofreading polymerase, Pfu, was identified in 1991 in Pyrococcus furiosus. This Pfu enzyme has a 3’ to 5’ exonuclease activity. As the DNA is amplified, the exonuclease removes mismatched nucleotides at the 3’ end of the strand. The correct nucleotide is then replaced, and DNA synthesis continues. The identification of incorrect nucleotide sequences is based on the binding affinity for the correct nucleoside triphosphate with the enzyme, where inefficient binding slows the synthesis and allows for the correct replacement. The proofreading activity of Pfu polymerase results in fewer errors in the final sequence compared to Taq DNA polymerase. In recent years, other proofreading enzymes have been identified, and modifications of the original Pfu enzyme have been made to further reduce the error rate during DNA amplification.

Reverse transcription PCR, or RT-PCR, allows the use of RNA as a template. An additional step allows the detection and amplification of RNA. The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The quality and purity of the RNA template are essential for the success of RT-PCR. The first step of RT-PCR is the synthesis of a DNA/RNA hybrid. Reverse transcriptase also has an RNase H function, which degrades the RNA portion of the hybrid. The single-stranded DNA molecule is then completed by the DNA-dependent DNA polymerase activity of the reverse transcriptase into cDNA. The efficiency of the first-strand reaction can affect the amplification process. From here on, the standard PCR procedure is used to amplify the cDNA. The possibility to revert RNA into cDNA by RT-PCR has many advantages, and it is primarily used for gene expression analysis. RNA is single-stranded and very unstable, which makes it challenging to work with. It commonly serves as a first step in qPCR, which quantifies RNA transcripts in a biological sample.

The PCR mentioned above techniques requires expensive thermocycling equipment to accurately ramp up and down chamber temperatures for the denaturation, annealing, and extension steps. A number of techniques have been developed that do not need such precise devices and can be performed in a simple water bath or even within the cells of interest. These techniques are collectively called isothermal amplification and work based on exponential, linear, or cascade amplification.

The best-known type of isothermal amplification is loop-mediated isothermal amplification, or LAMP. LAMP uses exponential amplification at 65⁰C to amplify template DNA or RNA. When performing LAMP, four to six primers complementary to regions of the target DNA are used with a DNA polymerase to synthesize new DNA. Two of these primers have complimentary sequences that recognize sequences in the other primers and bind them, allowing for a “loop” structure to form in the newly synthesized DNA that then aids primer annealing in subsequent rounds of amplification. LAMP can be visualized by multiple methods, including fluorescence, agarose gel electrophoresis, or colorimetry. The ease of visualizing and detecting the presence or absence of product by colorimetry and the lack of expensive equipment required made LAMP a suitable option for SARS-CoV-2 testing in areas where clinical lab testing was not readily available, or storage and transport of samples was not feasible, or in labs that did not previously have PCR thermocycling equipment.

The advantages of digital PCR have allowed for its use to be incorporated into several applications. The low amounts of RNA found in liquid biopsy samples make digital PCR ideal for these types of diagnostics, the analysis of single-cell samples, and detecting low amounts of pathogens during early infection. Digital PCR also allows for the detection of rare sequences and copy number variations since there is less background DNA in each partition to mask the specific amplification.

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