Navigation Links
PCR principles

In April, 1983, Kary Mullis took a drive on a moonlit California mountain road and changed the course of molecular biology. During that drive, he conceived the Polymerase Chain Reaction (PCR). As is shown in the figure, the reaction uses two oligonucleotide primers that hybridize to opposite strands and flank the target DNA sequence that is to be amplified. The elongation of the primers is catalyzed by a heat-stable DNA polymerase (such as Taq DNA Polymerase). A repetitive series of cycles involving template denaturation, primer annealing, and extension of the annealed primers by the polymerase results in exponential accumulation of a specific DNA fragment. The ends of the fragment are defined by the 5' ends of the primers. Because the primer extension products synthesized in a given cycle can serve as a template in the next cycle, the number of target DNA copies approximately doubles every cycle; thus, 20 cycles of PCR yield about a million copies of the target DNA. During the last decade, innovative researchers have continually updated the definition of "PCR applications", increasing the usefulness and scope of the technique. For instance:
Combining reverse transcription and PCR into the RT-PCR technique brought the benefits of PCR to analysis of RNA.
Using primers containing sequences that were not completely complementary to the template turned PCR into a tool for in vitro mutagenesis.
Replacing a single polymerase with a blend of a thermostable polymerase (Taq DNA Polymerase) and a proofreading polymerase (Pwo DNA Polymerase) made PCR an indispensable tool in the analysis and mapping of entire genomes by:
  • Extending the length of the sequence that could be amplified
  • Increasing the amount of PCR product
  • Providing higher fidelity during PCR
Using a Hot Start approach minimized the formation of primer-dimers during PCR.
Using short primers to produce a genomic fingerprint allowed analysis of organisms in which genomic sequences are largely unknown [e.g. Differ ential Display, Random Amplified Polymorphic DNA (RAPD)].
Introducing molecular tags, such as digoxigenin (DIG) or biotin-labeled dUTP into the PCR product, as it was amplified made PCR an invaluable tool for medical diagnostics. Such labeled PCR products may either be used as hybridization probes or be detected by use of capture probes. For instance, with PCR-generated DIG-labeled hybridization probes, it was possible to detect and quantify minute amounts of a pathogen.
Combining in situ hybridization with PCR (in situ PCR) made possible the localization of single nucleic acid sequences on one chromosome within an eukaryotic organism.
Extending PCR to the amplification of more than one sequence at a time (multiplex PCR) made it possible to compare two or more complex genomes, for instance to detect chromosomal imbalances.
The first automated system that combines amplification and detection of PCR and thereby minimizes handling time. A true diagnostic walk away system which guarantees integrity of the clinical results (COBAS AMPLICOR).
Combining online detection and continuous fluorescence monitoring (kinetic PCR) allowed more rapid quantification of PCR products (e.g. with LightCycler).
'"/>

Source:


Page: All 1 2 3 4

Related biology technology :

1. Praise for the principles of Peter
Post Your Comments:
*Name:
*Comment:
*Email:


(Date:2/23/2017)... and NEW YORK , Feb. ... Lumeon , a leading digital health company, and ... provider of telemedicine and remote patient monitoring, announce ... telemedicine reimbursements.  DN Telehealth maximizes ... in real-time, extending consultations beyond a physical clinical ...
(Date:2/23/2017)... 2017  MIODx announced today that it has ... technologies from the University of California, San Francisco ... monitor a patient for response to immune checkpoint ... second license extends the technology with a method ... have an immune-related adverse event (IRAE) from their ...
(Date:2/23/2017)... (PRWEB) , ... February 23, 2017 , ... ... (FDA) de novo clearance to begin marketing the SPEAC® System, the Brain Sentinel® ... adults at home or in healthcare facilities during periods of rest. A lightweight, ...
(Date:2/23/2017)... ... February 23, 2017 , ... The Greater Gift Initiative, Inc ... with Compass Research . GGI's mission is to advance global health and highlight ... in need in honor of each clinical trial volunteer. The vision of GGI is ...
Breaking Biology Technology:
(Date:2/8/2017)... 2017 Report Highlights ... The global synthetic-biology market reached nearly ... 2021, growing at a compound annual growth rate (CAGR) of ... the global markets for synthetic biology. - Analyses of global ... projections of compound annual growth rates (CAGRs) through 2021. - ...
(Date:2/8/2017)... Report Highlights The global biosurgery market should ... 2016 at a compound annual growth rate (CAGR) of ... An overview of the global market for biosurgery. - ... and 2016, and projections of compound annual growth rates ... the basis of product type, source, application, and region. ...
(Date:2/7/2017)... 7, 2017 Zimmer Biomet Holdings, Inc. (NYSE ... will present at the LEERINK Partners 6th Annual Global ... on Wednesday, February 15, 2017 at 10 a.m. Eastern ... presentation can be accessed at http://wsw.com/webcast/leerink28/zbh .  The ... via Zimmer Biomet,s Investor Relations website at http://investor.zimmerbiomet.com ...
Breaking Biology News(10 mins):