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The polymerase chain reaction (PCR) is a method widely used to make millions to billions of copies of a specific DNA sample rapidly, allowing scientists to amplify a very small sample of DNA sufficiently to enable detailed study. PCR was invented in 1983 by American biochemist Kary Mullis at Cetus Corporation. Mullis and biochemist Michael Smith, who had developed other essential ways of manipulating DNA, were jointly awarded the Nobel Prize in Chemistry in 1993.
A primer is a short, single-stranded nucleic acid used by all living organisms in the initiation of DNA synthesis. A synthetic primer may also be referred to as an oligo, short for oligonucleotide. DNA polymerase enzymes are only capable of adding nucleotides to the 3’-end of an existing nucleic acid, requiring a primer be bound to the template before DNA polymerase can begin a complementary strand. DNA polymerase adds nucleotides after binding to the RNA primer and synthesizes the whole strand. Later, the RNA strands must be removed accurately and replace them with DNA nucleotides forming a gap region known as a nick that is filled in using an enzyme called ligase. The removal process of the RNA primer requires several enzymes, such as Fen1, Lig1, and others that work in coordination with DNA polymerase, to ensure the removal of the RNA nucleotides and the addition of DNA nucleotides. Living organisms use solely RNA primers, while laboratory techniques in biochemistry and molecular biology that require in vitro DNA synthesis usually use DNA primers, since they are more temperature stable. Primers can be designed in laboratory for specific reactions such as polymerase chain reaction (PCR). When designing PCR primers, there are specific measures that must be taken into consideration, like the melting temperature of the primers and the annealing temperature of the reaction itself. Moreover, the DNA binding sequence of the primer in vitro has to be specifically chosen, which is done using a method called basic local alignment search tool (BLAST) that scans the DNA and finds specific and unique regions for the primer to bind.
Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA and amplification of specific DNA targets using polymerase chain reaction (PCR). It is primarily used to measure the amount of a specific RNA. This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR). Confusion can arise because some authors use the acronym RT-PCR to denote real-time PCR. In this article, RT-PCR will denote Reverse Transcription PCR. Combined RT-PCR and qPCR are routinely used for analysis of gene expression and quantification of viral RNA in research and clinical settings.
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. In this context, amplification refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as "PCR product."
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.
Thermodynamic databases contain information about thermodynamic properties for substances, the most important being enthalpy, entropy, and Gibbs free energy. Numerical values of these thermodynamic properties are collected as tables or are calculated from thermodynamic datafiles. Data is expressed as temperature-dependent values for one mole of substance at the standard pressure of 101.325 kPa, or 100 kPa. Both of these definitions for the standard condition for pressure are in use.
TaqMan probes are hydrolysis probes that are designed to increase the specificity of quantitative PCR. The method was first reported in 1991 by researcher Kary Mullis at Cetus Corporation, and the technology was subsequently developed by Hoffmann-La Roche for diagnostic assays and by Applied Biosystems for research applications.
SNP genotyping is the measurement of genetic variations of single nucleotide polymorphisms (SNPs) between members of a species. It is a form of genotyping, which is the measurement of more general genetic variation. SNPs are one of the most common types of genetic variation. An SNP is a single base pair mutation at a specific locus, usually consisting of two alleles. SNPs are found to be involved in the etiology of many human diseases and are becoming of particular interest in pharmacogenetics. Because SNPs are conserved during evolution, they have been proposed as markers for use in quantitative trait loci (QTL) analysis and in association studies in place of microsatellites. The use of SNPs is being extended in the HapMap project, which aims to provide the minimal set of SNPs needed to genotype the human genome. SNPs can also provide a genetic fingerprint for use in identity testing. The increase of interest in SNPs has been reflected by the furious development of a diverse range of SNP genotyping methods.
Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the random coil or single-stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its specific nucleotide sequence. DNA, when in a state where its two strands are dissociated, is referred to as having been denatured by the high temperature.
Multiplex ligation-dependent probe amplification (MLPA) is a variation of the multiplex polymerase chain reaction that permits amplification of multiple targets with only a single primer pair. It detects copy number changes at the molecular level, and software programs are used for analysis. Identification of deletions or duplications can indicate pathogenic mutations, thus MLPA is an important diagnostic tool used in clinical pathology laboratories worldwide.
Bisulfitesequencing (also known as bisulphite sequencing) is the use of bisulfite treatment of DNA before routine sequencing to determine the pattern of methylation. DNA methylation was the first discovered epigenetic mark, and remains the most studied. In animals it predominantly involves the addition of a methyl group to the carbon-5 position of cytosine residues of the dinucleotide CpG, and is implicated in repression of transcriptional activity.
The polymerase chain reaction (PCR) is a commonly used molecular biology tool for amplifying DNA, and various techniques for PCR optimization which have been developed by molecular biologists to improve PCR performance and minimize failure.
A primer binding site is a region of a nucleotide sequence where an RNA or DNA single-stranded primer binds to start replication. The primer binding site is on one of the two complementary strands of a double-stranded nucleotide polymer, in the strand which is to be copied, or is within a single-stranded nucleotide polymer sequence.
Loop-mediated isothermal amplification (LAMP) is a single-tube technique for the amplification of DNA and a low-cost alternative to detect certain diseases that was invented in 2000 at the University of Tokyo. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) combines LAMP with a reverse transcription step to allow the detection of RNA.
Primer Premier software combines design of primers for various PCR applications under one common platform. The software supports design of degenerate primers on alignments for amplifying a related set of nucleotide sequences for detecting many common traits amongst organisms and to determine heredity.
High Resolution Melt (HRM) analysis is a powerful technique in molecular biology for the detection of mutations, polymorphisms and epigenetic differences in double-stranded DNA samples. It was discovered and developed by Idaho Technology and the University of Utah. It has advantages over other genotyping technologies, namely:
A primer dimer (PD) is a potential by-product in the polymerase chain reaction (PCR), a common biotechnological method. As its name implies, a PD consists of two primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers. As a result, the DNA polymerase amplifies the PD, leading to competition for PCR reagents, thus potentially inhibiting amplification of the DNA sequence targeted for PCR amplification. In quantitative PCR, PDs may interfere with accurate quantification.
Multiplex polymerase chain reaction refers to the use of polymerase chain reaction to amplify several different DNA sequences simultaneously. This process amplifies DNA in samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. The primer design for all primers pairs has to be optimized so that all primer pairs can work at the same annealing temperature during PCR.
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 sequences from a sequenced genome or transcriptome.
Asymmetric PCR is a variation of PCR used to preferentially amplify one strand of the original DNA more than the other. The technique has applications in some types of sequencing and hybridization probing where having only one of the two complementary strands is required.
References
- ↑ http://www.premierbiosoft.com/netprimer/netprlaunch/Help/xnetprlaunch.html (although it says "requires Java plug-in 1.4 or later for Windows", further down the page it also shows they've tested it on Mac OS X)
- ↑ "Archived copy" (PDF). Archived from the original (PDF) on 2009-12-14. Retrieved 2009-01-07.
{{cite web}}: CS1 maint: archived copy as title (link) - ↑ Dieffenbach, C.W.; Dveksler, G.S. (2003). PCR Primer: A Laboratory Manual, Second Edition (2003) Paperback. Cold Spring Harbor Laboratory Press. p. 61. ISBN 9780879696542 . Retrieved 2015-05-17.
- ↑ pga.mgh.harvard.edu/primerbank/help.html
- ↑ "Archived copy" (PDF). Archived from the original (PDF) on 2009-01-10. Retrieved 2009-01-07.
{{cite web}}: CS1 maint: archived copy as title (link) - ↑ Callahan, Johnny D.; Wu, Shuenn-Jue L.; Dion-Schultz, Amanda; Mangold, Beverly E.; Peruski, Leonard F.; Watts, Douglas M.; Porter, Kevin R.; Murphy, Gerald R.; Suharyono, Wuryadi; King, Chwan-Chuen; Hayes, Curtis G.; Temenak, Joseph J. (2001). "Development and Evaluation of Serotype- and Group-Specific Fluorogenic Reverse Transcriptase PCR (Taq Man) Assays for Dengue Virus" (PDF). Journal of Clinical Microbiology. 39 (11): 4119–4124. doi:10.1128/JCM.39.11.4119-4124.2001. PMC 88496 . PMID 11682539.
- ↑ "PREMIER Biosoft".