Quantitative polymerase chain reaction, qPCR
Quantitative polymerase chain reaction (qPCR) also known as real time PCR is a PCR technique used for measuring a starting DNA concentration using PCR. qPCR requires the addition of a probe based fluorescent dye that intercalates with any dsDNA and the use of a fluorometer feature built into the thermocycler to measure that fluorescent output. With this fluorescent dye, the concentration of the DNA during the PCR reaction cycles is continuously detected via a fluorescent signal. The signal increases proportionally to the amount of product produced each cycle.
To determine the concentration of the starting template DNA the fluorescent signal throughout the reaction is compared to a standard curve of amplified DNA of a known starting concentration. The cycle in which the unknown DNA is detected compared to the standard curve can be used to determine the amount of starting material in your sample.
qPCR can also be used to quantitate RNA levels using reverse transcription-qPCR (RT-qPCR). The first step of this process requires RNA to be converted to cDNA using reverse transcription and the cDNA is subsequently quantified by qPCR. qPCR is used in a variety of applications including gene expression profiling, studying copy number variation, and molecular diagnostics.
Digital droplet PCR, ddPCR
Digital droplet PCR or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR where results can vary across replicates. ddPCR can be used to quantify DNA sequences that are rare, for example, rare alleles or mutations. ddPCR also does not require a reference or standard curve, which can be time consuming and challenging to get right. ddPCR uses a water-oil emulsion droplet technology that fractions the PCR reaction sample into approximately 20,000 droplets. Each droplet contains the material required for PCR amplification. Following the PCR reactions each droplet is analyzed by a droplet reader, which measures the fluorescence amplitude of each droplet. The fraction of fluorescent PCR-positive droplets is determined and then analyzed using Poisson statistics to determine the concentration of the original template DNA in the sample.
Addgene currently uses ddPCR for AAV titrating. You can learn all about ddPCR for AAV titration and find helpful tips and tricks in our blog post “Droplet Digital PCR for AAV Quantification.”
Multiplex PCR
Multiplex PCR, as the name implies, is a method in which multiple targets can be amplified in a single PCR experiment using multiple primers all in one PCR reaction. This is an extremely useful PCR method that can help save time and effort in the laboratory.
There are two main categories of multiplex PCR:
Single template PCR reaction - one template is amplified using several forward and reverse primer sets.
Multiple template PCR - multiple templates with different primer pairs that align to the target region of each template are used in one reaction.
Multiplex PCR is used commonly in disease or pathogen identification. Scientists can simultaneously detect several different pathogens in one specimen saving both time and effort. Scientists can also utilize multiplex qPCR to quantify the concentrations of multiple starting DNA templates in one sample. It is important to note however, that multiplex PCR is more complicated to develop and is often less sensitive than PCRs that use a single pair of primers, like those stated above. Multiplex PCR is also used in high throughput SNP genotyping, gene deletion analysis, and RNA detection.
A summary of the types of PCR
Type of PCR | Goal of the PCR method | Uses of PCR method |
End point PCR or just PCR | Amplify a region of interest | l Molecular Cloning l Site-directed mutagenesis l Molecular Diagnostics |
qPCR | Quantify starting template DNA concentration | l Gene expression profiling l Copy number variations l Quantify RNA levels l Molecular Diagnostics l NGS library prep QC |
ddPCR | Determine ultrasensitive and absolute nucleotide concentration of starting material | l AAV Titrations l Allelic Variations l Quantifying low abundance targets l Monitoring subtle changes in target DNA levels |
Multiplex PCR | Amplify multiple targets from one sample | l Disease or pathogen identification l SNP genotyping l Gene deletion Analysis l RNA detection |