A closed tube format for amplification and detection of DNA based on energy transfer
TL;DR: This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.
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Abstract: A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.
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References
•Book
PCR protocols : A guide to methods and applications
Michael A. Innis
- 01 Jan 1990
TL;DR: Basic Methodology: M.A. Innis and D.F. Frohman, RACE: Rapid Amplification of cDNA Ends, and RNA Processing: Apo-B.R. Kwok, Procedure to Minimuze PCR-Product Carry-Over.
13.1K
Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction.
Kary B. Mullis,Fred A. Faloona +1 more
TL;DR: A method whereby a nucleic acid sequence can be exponentially amplified in vitro is described in the chapter, and the possibility of utilizing a heat-stable DNA polymerase is explored so as to avoid the need for addition of new enzyme after each cycle of thermal denaturation.
6.6K
Molecular Beacons: Probes that Fluoresce upon Hybridization
Sanjay Tyagi,Fred Russell Kramer +1 more
TL;DR: Novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions that undergo a spontaneous conforma-tional change when they hybridize to their targets are developed.
4.9K
Detection of specific polymerase chain reaction product by utilizing the 5'----3' exonuclease activity of Thermus aquaticus DNA polymerase.
TL;DR: The 5'----3' exonuclease activity of the thermostable enzyme Thermus aquaticus DNA polymerase may be employed in a polymerase chain reaction product detection system to generate a specific detectable signal concomitantly with amplification.
3.2K
Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions
TL;DR: Because UDG does not react with dUTP, and is also inactivated by heat denaturation prior to the actual PCR, carry-over contamination of PCRs can be controlled effectively if the contaminants contain uracils in place of thymines.
1.1K