Nested Ligation-mediated Pcr
Nested Ligation-mediated Pcr - 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.
10:34
Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR
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Cited by 3 •
2013
A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor...
07:34
DNA Ligation Reactions
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2023
In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication. In the laboratory, DNA ligase is used during molecular cloning to join DNA fragments of inserts with vectors – carrier DNA molecules that will replicate target fragments in host organisms. This video provides an...
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.
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.
FAQs
The two primer types provide complementary targeting information. The adaptor-specific primer recognizes the known sequence attached to restriction-generated DNA ends, while the locus-specific primer anchors amplification at a genomic region already characterized by the investigator. Their combined use links an unknown neighboring fragment to a known genomic sequence, enabling selective analysis of regions that ordinary locus-only amplification may not readily access.
The second reaction uses internal primers positioned within the product generated during the first amplification. Any nonspecific products that lack the correct internal sequences are less likely to amplify efficiently in this round. This additional sequence requirement enriches fragments connected to the intended genomic region and helps reduce background, making the final product more suitable for analyzing nearby genomic DNA.
Restriction enzymes create DNA ends that can receive the known adaptors used for priming. Ligation therefore converts otherwise unknown fragment ends into recognizable sequences while preserving their association with genomic DNA. The resulting adaptor-genome junction supplies a defined priming site, which is essential for amplifying sequence adjacent to a known locus rather than relying on complete prior knowledge of the target.
A conventional PCR assay generally depends on primer-binding sites already known on both sides of a target. Nested ligation-mediated PCR instead combines a known genomic primer with an adaptor-derived primer, allowing investigation beyond a characterized sequence into an adjacent fragment. Its successive amplification rounds add specificity, which is particularly useful when the neighboring genomic sequence is unknown or when nonspecific products could interfere.
The workflow begins with genomic DNA cleavage by a restriction enzyme, followed by ligation of known adaptors to the resulting ends. An initial PCR then uses an adaptor-specific primer and a locus-specific primer. A second PCR uses internal primers within the first product. The final amplified fragments can be examined to characterize sequence adjacent to the known genomic region.
This approach is useful when investigators need to identify or characterize genomic DNA near a sequence that is already known. Applications include mapping insertion sites, examining DNA rearrangements, and studying sequence variation. Because the method uses successive amplification rounds and can work with limited genomic material, it supports targeted analysis when sample quantity or prior sequence information is constrained.