nested PCR

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nested PCR

amplification of an AMPLICON by using PRIMERS (nested primers) internal to the original primers used to generate that amplicon.


polymerase chain reaction. The amplification of a specific DNA sequence, termed target or template sequence, that is present in a complex mixture, by adding two or more short oligonucleotides, also called primers, that are specific for the terminal or outer limits of the template sequence. The template-primers mixture is subjected to repeated cycles of heating to separate (melt) the double-stranded DNA and cooling in the presence of nucleotides and DNA polymerase such that the template sequence is copied at each cycle. Thermostable polymerases such as those obtained from a hot springs bacterium Thermus aquaticus, commonly termed Taq polymerase, are used.
At the end of 20 to 30 such cycles, the amplified target sequence, which may have been present in as few as a single copy in the original mixture, can be readily detected, for example by electrophoresis and ethidium bromide staining in an argarose gel.

multiplex PCR
the detection of more than one template in a mixture by addition of more than one set of oligonucleotide primers.
nested PCR
the primers used in the first round of amplification are either both replaced (nested PCR) or only one is replaced (semi-nested PCR) for the second and subsequent cycles of amplification. Increases the sensitivity and specificity of the PCR.
quantitative PCR
a means for quantifying the amount of template DNA present in the original mixture. Usually achieved by the addition of a known amount of a target sequence that is amplified by the same primer set but can be differentiated, usually by size, at the end of the reaction.
real-time PCR
a method for the detection and quantitation of an amplified PCR product based on incorporation of a fluorescent reporter dye; the fluorescent signal increases in direct proportion to the amount of PCR product produced and is monitored at each cycle, 'in real time', such that the time point at which the first significant increase in the amount of PCR product correlates with the initial amount of target template.
reverse-transcriptase PCR (RT-PCR)
a reaction applied when the target sequence is RNA, such as viral RNA or messenger RNA. Reverse transcriptase that copies DNA from an RNA template is present in the first round.
References in periodicals archive ?
No false positive results were detected by LAMP or nested PCR among 15 blood samples from healthy individuals (a specificity of 100% for both) The minimum detection limit of LAMP was 5 parasites/[micro]l compared to 2 parasites/[micro]l by PCR and 20 parasites/[micro]l by microscopy in case of P falciparum, 10 parasites/[micro]l compared to 2 parasites/ [micro]l by PCR and 20 parasites/[micro]l by microscopy in case of P vivax and 20 parasites/[micro]l compared to 2 parasites/ pl by PCR and 30 parasites/[micro]l by microscopy in case of P malariae.
The method involved restriction enzyme analysis of nested PCR products obtained with primers encoding for the 65-kDa protein, which was common to all mycobacteria.
We designed degenerate primers for nested PCR screening on the basis of a multiple sequence alignment of the NS1 gene from human BuV, WUHARV parvovirus and MpBuV as follows: BuV-F1 (position 190-215 in MpBuV genome, 5'-TCAAWRTMACCTGGAAAGACrACAGA-3') and BuV-R1 (position 1503 1534, 5'-TCATTGGTTGTCATKAYWACTGGAGTTGGTTC-3') for the first PCR round, and BuV-F2 (position 980-1006, containing an equimolar mixture of 5'-AGAAAAATGGATGCTCCAAGATCCAGA-3' and 5'-AGAAAAATGGATGCTTGGTGAWCCWGA-3') and BuV-R2 (position 1444 1465, 5'-ATTGCTTGGCCACTCATGATKG-3') for the second PCR round.
The team working on Xpert MTB/RIF Ultra intends to capitalise on Cepheid's cartridge-based nested PCR amplification capability to amplify patient DNA samples to a level that can be more accurately detected.
La sensibilidad analitica de la Nested PCR fue altamente eficiente y confirmada aun mas cuando fue evaluada mediante la prueba de KruskalWallis, la misma que determino que el mejor tratamiento de [T.
Nested PCR of ITS-rDNA was used for detecting and identifying Leishmania species in sandflies.