Nested Pcr Amplification
Nested Pcr Amplification - Related Videos
03:20
Nested-PCR to Detect a Specific Viral Genomic Sequence
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2025
This video describes nested polymerase chain reaction, a technique that consists of two sequential PCR amplification processes using two primer sets. The first set of primers is intended to anneal to sequences upstream of the second set, resulting in selective amplification of specific gene sequences. This PCR is more sensitive and specific than a normal PCR and is widely used as a detection technique for various diseases.
09:44
Rapid Amplification of cDNA Ends
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2023
Source: Pablo Sanchez Bosch2, Sean Corcoran2 and Katja Brückner1,2,3 1Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research 2Department of Cell and Tissue Biology, 3Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA, USA Rapid Amplification of cDNA Ends (RACE) is a technique that allows amplification of full-length cDNA from mRNA by extending to the 3’ or 5’ end, even without prior knowledge of the sequence (Frohman et al.,...
11:58
Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA
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Cited by 14 •
2014
Linear-amplification mediated (LAM)-PCR is a method developed to identify the exact positions of integrating viral vectors in the genome. The technique has evolved to be the superior method to study clonal dynamics in gene therapy patients, biosafety of novel vector technologies, T-cell diversity, cancer stem cell models, etc.
07:20
Detecting the Lyme Disease Spirochete, Borrelia Burgdorferi, in Ticks Using Nested PCR
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Cited by 21 •
2018
Nested PCR is a sensitive, specific, and straightforward technique that can be applied to tick DNA extracts to probe for Borrelia burgdorferi, the causative agent of Lyme disease. The initial PCR experiment uses gene-specific primers to generate long amplicons, which then become templates for a subsequent reaction using internal primers.
10:12
Protein Misfolding Cyclic Amplification of Prions
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Cited by 7 •
2012
Protein misfolding cyclic amplification (PMCA) is an in vitro assay for the study of prion conversion and strain and species barriers. It can also be used as a prion detection assay.
FAQs
The inner primers improve selectivity because they must recognize a sequence located within the product generated by the outer primer pair. A nonspecific first-round product is less likely to support the correct second-round amplification unless it also contains the matching internal target region. This two-level recognition can make the final signal more closely associated with the intended DNA sequence.
Outer and inner primers contribute different targeting layers rather than serving interchangeable roles. The outer pair first defines a broader region containing the sequence of interest, whereas the inner pair focuses amplification on a smaller internal segment. This arrangement explains why nested PCR amplification can distinguish the intended target from products generated through less selective conventional PCR.
Nested PCR amplification is particularly useful when the target occurs at low abundance or when conventional PCR yields weak or nonspecific signals. The first round can enrich material that may be difficult to detect directly, and the second round then applies additional sequence selection. The resulting gain in detectability and specificity supports confirmation of microbial or immune-related DNA targets.
Each round increases the amount of amplified DNA available for subsequent detection, so carryover from an earlier reaction can be mistaken for a true target signal. This creates a heightened risk of false-positive results. In immunology and infection studies, contamination control is therefore central to interpreting pathogen-associated or immune-related sequences as evidence of the biological target.
Planning begins with an outer primer pair for the first amplification, followed by a second reaction using inner primers that bind within the initial amplicon. The workflow therefore depends on preserving the first-round product as the template for the second round while preventing unwanted carryover between reactions. The design links target enrichment directly to sequence-specific confirmation.
It can support identification of microbial nucleic acids, confirmation of pathogen-associated sequences, and analysis of immune-related genetic targets. These uses are especially relevant when a conventional PCR result is weak or contains nonspecific products. Results can provide evidence that a selected DNA sequence is present, but interpretation must account for the possibility of false positives from contamination.