Inverse Pcr Analysis
Inverse Pcr Analysis - Related Videos
10:54
Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR
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Cited by 2 •
2019
This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.
12:04
RNA Analysis of Environmental Samples Using RT-PCR
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2023
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona Demonstrating Author: Bradley Schmitz Reverse transcription-polymerase chain reaction (RT-PCR) involves the same process as conventional PCR — cycling temperature to amplify nucleic acids. However, while conventional PCR only amplifies deoxyribonucleic acids (DNA), RT-PCR enables the amplification of ribonucleic acids (RNA) through the formation of complementary DNA (cDNA). This enables RNA-based organisms...
06:30
Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis
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Cited by 34 •
2014
PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.
08:42
An Inverse Analysis Approach to the Characterization of Chemical Transport in Paints
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Cited by 2 •
2014
In this paper, a procedure for quantifying the mass transport parameters of chemicals in various materials is presented. This process involves employing an inverse-analysis based diffusion model to vapor emission profiles recorded by real-time, mass spectrometry in high vacuum.
12:53
Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow
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Cited by 5 •
2017
This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.
FAQs
Outward-facing primers extend away from the known sequence and toward unknown DNA after circularization. When the ligated fragment contains the target region and its flanking sequence, amplification crosses the newly formed junction, producing a product that connects known primer-binding sites with previously uncharacterized DNA. This design converts unknown adjacency into a sequenceable PCR product.
Restriction digestion determines the DNA fragment boundaries available for circularization. Ligation then joins the resulting fragment ends, creating a circular template in which sequences originally separated in genomic DNA become connected through a junction. The target fragment must include the known region and neighboring unknown DNA so that the outward-facing primers occupy an amplifiable arrangement.
Sequencing the PCR product reveals DNA adjacent to the known region rather than merely confirming amplification of that known sequence. Researchers can use the recovered sequence to map a transgene or insertion site, examine a genomic rearrangement, or identify nearby regulatory or genomic regions. Interpretation therefore links a defined sequence to its previously unknown genomic context.
A typical workflow begins with genomic DNA digestion, followed by ligation to circularize the resulting fragments. Primers are designed within the known region with an outward orientation, and PCR amplifies across the circularized junction. The resulting product is then sequenced to recover flanking DNA. These stages provide fragmentation, circular template formation, selective amplification, and sequence-based identification.
Inverse PCR analysis is useful when a researcher knows one DNA segment but lacks its surrounding sequence. During molecular characterization of transgenes or other insertions, locating the integration site requires connecting the defined sequence with neighboring genomic DNA. The method provides a route from a known construct-associated region to its unknown genomic context for mapping and characterization.
The recovered flanking sequence can address several mapping and characterization questions. It may identify where an insertion lies, help examine a genomic rearrangement, or recover adjacent regulatory or genomic regions. These applications connect a molecular event with surrounding sequence information, making the approach relevant to genetic mapping, genome analysis, and molecular characterization in biology.