Quantitative Methylation-specific Pcr
Quantitative Methylation-specific Pcr - Related Videos
09:05
Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics
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2020
Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.
21:24
Methylated DNA Immunoprecipitation
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Cited by 43 •
2009
This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).
12:34
DNA Methylation: Bisulphite Modification and Analysis
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Cited by 75 •
2011
The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.
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.
05:59
Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos
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Cited by 24 •
2015
A method for fluorescent staining of fixed biological material with the specific DNA label methyl green is described. Methyl green is used in a diluted aqueous solution and is very resistant to photobleaching. Its far-red emission allows for deep specimen imaging, making it particularly adequate for whole embryos.
FAQs
Bisulfite treatment creates the sequence difference that qMSP exploits. Unmethylated cytosines are converted to uracil, whereas methylated cytosines remain unchanged, so the resulting DNA carries methylation-dependent sequence patterns. Primers designed for these patterns can preferentially identify methylated regions, linking amplification to the methylation state of a selected genomic locus.
Because the primers are methylation-specific, qMSP does not survey methylation across the entire genome; it interrogates selected genomic regions. That targeted design allows investigators to focus on genes or regulatory regions in a sample whose methylation may be relevant to gene regulation or cancer-associated epigenetic change.
Fluorescent real-time PCR adds a quantitative dimension to the assay. Rather than only indicating whether a methylation-specific sequence is detectable, the method tracks amplification through fluorescence and produces a measurement for the targeted methylated sequence. This output supports comparisons among samples, such as tumor and normal DNA, when the same genomic target is examined.
The workflow preserves the distinction created during bisulfite conversion. DNA is first subjected to the conversion treatment, then analyzed with primers matched to methylated sequence patterns, and finally monitored during real-time PCR through fluorescence. Keeping these stages linked is important because the final quantitative result depends on the converted template and the selected target.
In cancer research, qMSP can examine methylation in tumor suppressor genes and compare the results with patterns in normal samples. Such comparisons help reveal cancer-associated epigenetic differences at selected loci. The assay therefore supports focused investigations of how altered methylation relates to tumor biology without requiring a genome-wide methylation survey.
Because it produces quantitative measurements, qMSP can contribute to biomarker development and studies of disease progression, diagnosis, prognosis, and treatment response. Its value in these settings comes from comparing methylation levels at defined targets across relevant samples. The resulting measurements help connect a specific epigenetic pattern with a cancer-related research question.