In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

Nat Methods. 2004 Dec;1(3):227-32. doi: 10.1038/nmeth723. Epub 2004 Nov 18.

Abstract

Methods are needed to study single molecules to reveal variability, interactions and mechanisms that may go undetected at the level of populations of molecules. We describe here an integrated series of reaction steps that allow individual nucleic acid molecules to be detected with excellent specificity. Oligonucleotide probes are circularized after hybridization to target sequences that have been prepared so that localized amplification reactions can be initiated from the target molecules. The process results in strong, discrete detection signals anchored to the target molecules. We use the method to observe the distribution, within and among human cells, of individual normal and mutant mitochondrial genomes that differ at a single nucleotide position.

Publication types

  • Comparative Study
  • Evaluation Study
  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • DNA Mutational Analysis / methods
  • DNA Probes / chemistry
  • DNA Probes / genetics*
  • DNA, Mitochondrial / analysis
  • DNA, Mitochondrial / genetics*
  • Gene Targeting / methods*
  • Genetic Testing / methods
  • Genotype
  • Humans
  • In Situ Hybridization, Fluorescence / methods*
  • Mitochondrial Diseases / classification
  • Mitochondrial Diseases / diagnosis
  • Mitochondrial Diseases / genetics*
  • Nucleic Acid Amplification Techniques / methods*
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Alignment / methods
  • Sequence Analysis, DNA / methods*

Substances

  • DNA Probes
  • DNA, Mitochondrial