In silico PCR refers to computational tools used to calculate theoretical
polymerase chain reaction (PCR)
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
results using a given set of
primers (
probes) to amplify
DNA sequences from a sequenced
genome
In the fields of molecular biology and genetics, 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 ge ...
or
transcriptome
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...
.
These tools are used to optimize the design of primers for target DNA or
cDNA
In genetics, complementary DNA (cDNA) is DNA synthesized from a single-stranded RNA (e.g., messenger RNA (mRNA) or microRNA (miRNA)) template in a reaction catalyzed by the enzyme reverse transcriptase. cDNA is often used to express a speci ...
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 three dimensional conformational isomerism, form of ''local segments'' of proteins. 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
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification (molecular biology), amplification or DNA replication, replication events. It can be formed artificially, using various methods including ...
, simulation of
electrophoretic
Electrophoresis, from Ancient Greek ἤλεκτρον (ḗlektron, "amber") and φόρησις (phórēsis, "the act of bearing"), is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric fi ...
mobility, identification of
open reading frames
In molecular biology, open reading frames (ORFs) are defined as spans of DNA sequence between the start and stop codons. Usually, this is considered within a studied region of a prokaryotic DNA sequence, where only one of the six possible readin ...
, 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 United States 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) website. On the other hand, FastPCR,multiplex PCR Multiplex polymerase chain reaction (Multiplex PCR) refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously (as if performing many separate PCR reactions all together in one reaction). This process ...
. VPCRUCSC Genome Browser
The UCSC Genome Browser is an online and downloadable genome browser hosted by the University of California, Santa Cruz (UCSC). It is an interactive website offering access to genome sequence data from a variety of vertebrate and invertebrate spec ...
offer
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
Webtools for PCR, qPCR, ''in silico'' PCR and oligonucleotides
''In silico'' simulation of molecular biology experiments
Nucleic acids
Bioinformatics