Single-nucleotide sequence discrimination in situ using padlock probes.
Standard fluorescence in situ hybridization (FISH) techniques using cloned probes are limited in their ability to distinguish between closely similar DNA sequences because long hybridization probes are not detectably destabilized by single mismatched base pairs. This problem has been addressed by using short allele-specific oligonucleotide probes whose hybridization to target sequences is more sensitive to mismatches. This revised and expanded unit presents protocols for discrimination between closely similar DNA sequences in situ. The discussion of probe synthesis has been greatly expanded and an Alternate Protocol 1 added for enzymatic probe ligation at low probe concentration. A new Support Protocol describes enzymatic probe synthesis.
Nilsson
M
M
Uppsala University, Uppsala, Sweden.
Landegren
U
U
Antson
D O
DO
D000483:Alleles; D000818:Animals; D004247:DNA / Q000032:analysis; D011088:DNA Ligases / Q000378:metabolism; D015342:DNA Probes / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000032:analysis
D015342:DNA Probes; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004247:DNA; D011088:DNA Ligases