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Related Concept Videos

Labeling DNA Probes03:31

Labeling DNA Probes

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
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Updated: May 20, 2025

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain
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Investigating Single-Molecule Molecular Inversion Probes for Medium-Scale Targeted DNA Methylation Analysis.

Roy B Simons1, Hieab H H Adams2, Manfred Kayser1

  • 1Department of Genetic Identification, Erasmus MC, University Medical Center Rotterdam, 3015 GD Rotterdam, The Netherlands.

Epigenomes
|March 26, 2025
PubMed
Summary
This summary is machine-generated.

Single-molecule molecular inversion probes (smMIPs) show promise for analyzing CpG methylation with low DNA input. However, current smMIP designs lack specificity and uniformity for bisulfite-converted DNA.

Keywords:
DNA methylationage predictioncaptureepigeneticspadlock probessingle-molecule molecular inversion probesunique molecular identifiers

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Area of Science:

  • Epigenetics
  • Molecular Biology
  • Biotechnology

Background:

  • CpG methylation is a key epigenetic biomarker in clinical and forensic applications.
  • A cost-effective, sensitive method for medium-scale CpG analysis with low DNA input (<10 ng) is needed.
  • Single-molecule molecular inversion probes (smMIPs) are explored for simultaneous analysis of hundreds of CpGs.

Purpose of the Study:

  • To investigate the utility of smMIPs for analyzing hundreds of user-defined CpGs.
  • To develop a novel smMIP design tool (Locksmith) for bisulfite-converted DNA.
  • To optimize smMIP capture conditions for sensitive methylation analysis.

Main Methods:

  • Developed a novel smMIP design tool (Locksmith) tailored for bisulfite-converted DNA.
  • Optimized smMIP capture through single-probe testing and full panel evaluation.
  • Varied hybridization/elongation temperatures, smMIP/DNA amounts, dNTP concentration, and elongation time.

Main Results:

  • Capture efficiency varied significantly based on probe sequence, leading to heterogeneous CpG coverage.
  • Robust methylation detection was achieved for CpGs with ≥20X coverage, comparable to EPIC microarray analysis (r=0.96).
  • Low specificity and uniformity were observed across the tested smMIP panel.

Conclusions:

  • Current smMIP designs exhibit low specificity and uniformity with bisulfite-converted DNA.
  • smMIPs, in their present form, are not suitable for analyzing the reduced complexity of bisulfite-converted DNA.
  • Further optimization of smMIP design and protocols is required for effective epigenetic analysis.