In silico PCR refers to computational tools used to calculate theoretical
polymerase chain reaction (PCR) results using a given set of
primers (
probes) to amplify
DNA
Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of al ...
sequences from a sequenced
genome
A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...
or
transcriptome.
These tools are used to optimize the design of primers for target DNA or
cDNA
In genetics, complementary DNA (cDNA) is DNA that was reverse transcribed (via reverse transcriptase) from an RNA (e.g., messenger RNA or microRNA). cDNA exists in both single-stranded and double-stranded forms and in both natural and engin ...
sequences.
Primer optimization has two goals: efficiency and selectivity. Efficiency involves taking into account such factors as GC-content, efficiency of binding, complementarity,
secondary structure
Protein secondary structure is the local spatial conformation of the polypeptide backbone excluding the side chains. The two most common Protein structure#Secondary structure, secondary structural elements are alpha helix, alpha helices and beta ...
, and
annealing and melting point (Tm). Primer selectivity requires that the primer pairs not fortuitously bind to random sites other than the target of interest, nor should the primer pairs bind to conserved regions of a gene family. If the selectivity is poor, a set of primers will amplify multiple products besides the target of interest.
The design of appropriate short or long primer pairs is only one goal of PCR product prediction. Other information provided by ''in silico'' PCR tools may include determining primer location, orientation, length of each
amplicon, simulation of
electrophoretic mobility, identification of
open reading frames, and links to other web resources.
Many software packages are available offering differing balances of feature set, ease of use, efficiency, and cost.
[Primer-BLAST]
is widely used, and freely accessible from the National Center for Biotechnology Information
The National Center for Biotechnology Information (NCBI) is part of the National Library of Medicine (NLM), a branch of the National Institutes of Health (NIH). It is approved and funded by the government of the United States. The NCBI is lo ...
(NCBI) website. On the other hand, FastPCR,free software
Free software, libre software, libreware sometimes known as freedom-respecting software is computer software distributed open-source license, under terms that allow users to run the software for any purpose as well as to study, change, distribut ...
that outputs in-silico PCR products from primer sets provided in a FASTA file. It is fast (through use of a genome's FM-index) and can account for primer melting temperature and tolerated edit distances between primers and hit locations on the genome. VPCR
isPCR
which provides graphical as well text-file output to view PCR products on more than 100 sequenced genomes.
A primer may bind to many predicted sequences, but only sequences with no or few mismatches (1 or 2, depending on location and nucleotide) at the 3' end of the primer can be used for polymerase extension. The last 10-12 bases at the 3' end of a primer are sensitive to initiation of polymerase extension and general primer stability on the template binding site. The effect of a single mismatch at these last 10 bases at the 3' end of the primer depends on its position and local structure, reducing the primer binding, selectivity, and PCR efficiency.[
]
References
{{reflist, 30em
External links
* ttp://insilico.ehu.es/ ''In silico'' simulation of molecular biology experiments
Nucleic acids
Bioinformatics