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Related Experiment Videos

Genomic sequencing by ligation-mediated PCR

G P Pfeifer1, A D Riggs

  • 1Department of Biology, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.

Molecular Biotechnology
|June 1, 1996
PubMed
Summary

Genomic sequencing using ligation-mediated PCR (LMPCR) allows detailed DNA studies. This method provides high-quality results from small DNA amounts, enabling analysis of methylation and protein interactions.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biochemistry

Background:

  • Mammalian genome complexity limits genomic sequencing applications.
  • In vivo DNA methylation and protein-DNA interactions are crucial biological processes.
  • Sensitive methods are needed for detailed genomic analysis.

Purpose of the Study:

  • To present a detailed protocol for genomic sequencing using ligation-mediated PCR (LMPCR).
  • To demonstrate the utility of LMPCR for studying DNA methylation, in vivo footprinting, and DNA adduct mapping.
  • To highlight the adaptability and reproducibility of LMPCR for complex mammalian genomes.

Main Methods:

  • Genomic sequencing utilizing ligation-mediated PCR (LMPCR).
  • Protocol detailed for high-quality, reproducible sequence ladder generation.
  • Adaptation of LMPCR for methylation analysis, in vivo footprinting, and DNA adduct mapping.

Main Results:

  • LMPCR generates high-quality, reproducible sequence ladders from minimal DNA input (1 microgram).
  • The method is sensitive and applicable to uncloned mammalian DNA.
  • Successful adaptation for various in vivo genomic studies was demonstrated.

Conclusions:

  • Ligation-mediated PCR is a powerful and sensitive technique for genomic sequencing.
  • LMPCR overcomes limitations posed by mammalian genome complexity.
  • The protocol facilitates diverse applications in molecular biology and genomics research.

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