PCR allows millions of copies of a known target sequence to be made, and it is the most important technique in Molecular Biology
Real-time PCR or quantitative PCR is a PCR-based technique used to quantify the relative expression of genes or the absolute quantification of nucleic acids
A modern quantitative PCR method that allows to measure the amounts of DNA and RNA present in a sample
This Oxford Nanopore technology provides long readings in real time
A very high throughput massively parallel sequencing method
Sequencing of long DNA molecules in real time
Standard PCR is a basic but powerful technique due to its large number of applications.
Among them are the amplification of specific DNA or cDNA sequences for their subsequent cloning and construction of libraries or for their sequencing, the molecular diagnosis of diseases, the marker-assisted selection, among others.
Likewise, PCR is the basis for numerous molecular marker techniques such as microsatellite markers that can be used for unequivocal identification of varieties, characterization of germplasm collections, or studying the genetic stability of micropropagated plants.
qPCR differs from standard or conventional PCR in its quantitative nature.
Therefore, it is a widely used method to estimate the relative expression of genes.
For example, if you want to know the expression levels of one or more specific genes at different stages of development, organs or physiological conditions, this technique can be very useful.
It can also be used as a diagnostic method for viral, bacterial and fungal diseases.
Droplet Digital PCR is a similar method to qPCR as it uses the same reaction components, but differs in the way the amount of amplified product is measured in absolute terms.
In this technique, the PCR reaction is separated into thousands of drops in the order of nanoliters in an oil-water emulsion such that in each drop there is a single target molecule or a few.
Then a device measures each drop for the presence (1) or absence (0) of fluorescence signal, hence the name digital, then with statistical processing the amount of DNA molecules in the sample is determined.
This technique does not replace qPCR in all applications but is more appropriate for certain studies such as the detection of mutations, SNPs, number of copies of a gene within a genome and to detect small changes in the expression of genes.
MinION is a portable device that is capable of sequencing entire genomes in real time.
The basic principle of its operation is based on passing a nucleic acid molecule through a protein nanopore and monitoring electrical current changes in real time due to the type of nucleic acid.
Nanopore technology makes it possible to obtain much longer fragments than those obtained by the rest of the sequencing technologies, which allows a better and easier assembly of the genomes.
Its ability to generate data in real time allows instant identification of organisms in complex samples (metagenomics).
Illumina uses a four-color synthesis sequencing system, in which the incorporation of a reversible terminator nucleotide generates a fluorescent signal detected by a high-sensitivity camera for each base.
It offers various platforms with different performance such as MiniSeq (7.5 Gb), NextSeq (120-300Gb), HiSeq (125-1500 Gb) and NovaSeq 6000 (134–6000 Gb).
Illumina can generate many millions of highly accurate readings making it faster and cheaper than other available methods.
This technique uses what is called Zero Mode Waveguides (ZMW), a nanophotonic confining structure (approximately 70 nm in diameter and 100 nm deep). The sequencing is done on a chip that contains many ZMWs. At the bottom of each of these holes a DNA polymerase is attached with a single single-stranded DNA molecule. Each of the four nitrogenous bases in DNA is labeled with a different fluorophore. Thus, when a nucleotide is incorporated into the incipient chain, the fluorophore is released and a signal is observed that is collected in a detector.
The great advantage of PacBio is its ability to produce long reads, which makes it very suitable for de novo assembly of genomes. Compared to Illumina, it still has a lower performance and is not very suitable for RNA-seq. These techniques complement each other.