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Related Concept Videos

Real Time RT-PCR02:57

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Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is...
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Related Experiment Video

Updated: Aug 22, 2025

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
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Quantitative analysis of DNA methylation using sequence-specific, real-time loop-mediated isothermal amplification.

Yao Fu1, Li Zhang1, Yu Xing1

  • 1Department of Forensic Medicine, Chongqing Medical University, Chongqing, 400016, China.

Analytica Chimica Acta
|November 11, 2022
PubMed
Summary

This study introduces a novel method for accurately quantifying DNA methylation using a specialized probe in loop-mediated isothermal amplification (LAMP). This technique offers a sensitive and accessible tool for epigenetic diagnostics, particularly in resource-limited settings.

Keywords:
DNA methylationLoop-mediated isothermal amplificationReal-time fluorescence probesSequence specificity

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Area of Science:

  • Epigenetics and Molecular Diagnostics
  • Biotechnology and Biosensing

Background:

  • Anomalous DNA methylation is linked to genetic and tumor diseases, necessitating accurate methylation level determination for epigenetic diagnostics.
  • Existing loop-mediated isothermal amplification (LAMP) methods for methylation detection often lack systematic design principles and ambient applicability.

Purpose of the Study:

  • To develop a sequence-specific, real-time LAMP method for quantitative DNA methylation analysis.
  • To design and optimize a novel oligonucleotide dimer (PSS probe) for simultaneous primer, sense probe, and signal probe functions in LAMP.
  • To assess the method's sensitivity and applicability in detecting cancer-related gene methylation.

Main Methods:

  • Design of a multifunctional oligonucleotide dimer (PSS probe) for real-time LAMP-based DNA methylation detection.
  • Systematic investigation and optimization of the PSS probe's performance.
  • Quantitative analysis of synthetic methylated DNA and detection of KAZN gene methylation in colorectal cancer cells.

Main Results:

  • The developed method accurately quantifies as low as 0.1% synthetic methylated DNA amidst excess unmethylated DNA.
  • A higher methylation level of the KAZN gene was detected in colorectal cancer cells.
  • Stable housekeeping gene β-actin was utilized as an endogenous control for accurate quantification.

Conclusions:

  • The PSS probe-based LAMP method provides a sensitive and quantitative approach for DNA methylation analysis.
  • This technique is suitable for resource-limited environments, offering a valuable option for epigenetic diagnostics.
  • The method holds significant potential for the diagnosis of methylation-related diseases, including cancer.