Qpcr Deletion Screening
Qpcr Deletion Screening - Related Videos
11:24
Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs
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Cited by 8 •
2017
Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.
11:31
Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells
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Cited by 23 •
2016
Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).
09:57
Quantifying Environmental Microorganisms and Viruses Using qPCR
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2023
Source: Laboratories of Dr. Ian Pepper and Dr. Charles Gerba - The University of Arizona Demonstrating Author: Bradley Schmitz Quantitative polymerase chain reaction (qPCR), also known as real-time PCR, is a widely-used molecular technique for enumerating microorganisms in the environment. Prior to this approach, quantifying microorganisms was limited largely to classical culture-based techniques. However, the culturing of microbes from environmental samples can be particularly challenging, and...
09:21
Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay
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Cited by 3 •
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The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.
12:32
EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR
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Cited by 13 •
2016
Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.
FAQs
The assay compares amplification of the suspected deleted region with amplification of a reference locus from the same genomic DNA sample. Fluorescence is monitored during PCR, and the resulting cycle threshold values show the relative amplification behavior of both loci. A reduced target signal relative to the reference supports loss of the defined DNA region rather than an unchanged genome.
The reference locus provides an internal comparison for the target sequence. Because both regions are assessed from the same genomic DNA preparation, their cycle threshold values can be evaluated together rather than relying on target amplification alone. This comparison helps identify reduced target copy number and makes the result more informative for distinguishing modified samples from unchanged ones.
A sample containing both modified and unchanged genomes may produce an intermediate target signal relative to the reference locus. Such a result can indicate that the deletion is present in only part of the tested population rather than uniformly across all cells. This makes the approach useful for screening cell lines or other samples before more detailed genotyping.
Researchers first obtain genomic DNA from the sample, then amplify the sequence targeted for deletion together with a reference locus. Fluorescence is measured as amplification proceeds, and cycle threshold values are compared between the two regions. The relative result is then used to classify samples as consistent with a deletion, unchanged, or potentially mixed, depending on the observed target copy number.
The method can be applied to genomic DNA from embryos, cell lines, or tissue samples. This range supports screening at different stages of developmental biology research, from identifying engineered changes in embryos to checking established cell populations or collected tissues. In each case, the analysis focuses on whether the defined target region remains present relative to the reference locus.
qPCR deletion screening is useful when researchers need a rapid and sensitive way to screen many candidate samples for a defined deletion. It can identify samples that warrant further characterization, while sequencing or other genotyping methods can provide complementary information about the genetic change. In developmental studies, this combination helps connect engineered deletions with embryo, cell, or tissue analyses.