Single-nucleotide sequence discrimination in situ using padlock probes.

DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation. This mechanism has been exploited to distinguish DNA sequence variants in situ using so-called padlock probes. Padlock probes are linear oligonucleotides with target-complementary sequences at both ends, and an on-target-complementary segment in between. The end sequences are brought next to each other upon hybridization to the target DNA sequence, and if the ends are perfectly matched to the target sequence, they can be joined by a DNA ligase. Padlock probes detect target sequences with very high specificity, because both probe segments must hybridize to the target for circularization to occur. This unit presents a protocol for discrimination between closely similar DNA sequences in situ using padlock probes. A discussion of methods for greatly amplifying the signal from circularized probes is also included.DNA ligases are very sensitive to mismatches at the DNA ends to be joined through ligation.