In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes.
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.
Larsson
Chatarina
C
Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, Se-75185 Uppsala, Sweden.
Koch
Jørn
J
Nygren
Anders
A
Janssen
George
G
Raap
Anton K
AK
Landegren
Ulf
U
Nilsson
Mats
M
D004252:DNA Mutational Analysis / Q000379:methods; D015342:DNA Probes / Q000737:chemistry / Q000235:genetics*; D004272:DNA, Mitochondrial / Q000032:analysis / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D005820:Genetic Testing / Q000379:methods; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D028361:Mitochondrial Diseases / Q000145:classification / Q000175:diagnosis / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
D015342:DNA Probes; D004272:DNA, Mitochondrial