nested PCR


Also found in: Acronyms.

nested PCR

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

PCR1

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 ?
The opening of predispensed reaction mixes must be performed with great care, particularly between nested PCR steps.
A nested PCR was performed on both samples at optimal annealing temperatures with 40 cycles in the first and 30 cycles in the second PCR step.
We first confirmed by specific nested PCR the presence of HPyV9 in the skin swab specimen of the index patient in which the virus had been identified by HTS.
Among various PCR methods available for identification of human malaria parasite the nested PCR is considered as one of the most sensitive methods (11).
The analysis of msp-2 gene performed by nested PCR by using allelic family-specific primers generally confirmed the results of PCR-RFLP with few differences.
Forward and reverse primers were designed to amplify a 751 bp region of the gB gene, followed by a nested PCR with 5 forward primers (specific for five of the gB genotypes, gB1 to gB5) and a common reverse primer.
The FDA either did not read the contents of the petition before writing its decision or failed to keep up with the scientific advances since 1988 when the Digene HC2 HPV test was approved for marketing because neither the HPV DNA nested PCR device submitted for its review, nor the recommended accurate HPV genotyping by direct automated DNA sequencing for validation uses any probes at all," said Dr.
Nested PCR products were sequenced to confirm SFTSV sequences.
Cycling conditions for the semi-nested PCR were, 45 cycles: 94[degrees]C for 3 min, 94[degrees]C for 30 sec, 56[degrees]C for 30 sec, 60[degrees]C for 45 sec, 65[degrees]C for 15 min while that of nested PCR were, 35 cycles: 94[degrees]C for 3 min, 94[degrees]C for 30 sec, 56[degrees]C for 30 sec, 60[degrees]C for 45 sec, and finally 65[degrees]C for 10 min.
The aim of the present study was to compare the efficiency of PCR-based RFLP and multiplex nested PCR for gB gene of HCMV to determine their genotype in clinical specimens from patients with HCMV.