PCR-generated padlock probes detect single nucleotide variation in genomic DNA.
Circularizing oligonucleotide probes, so-called padlock probes, have properties that should prove valuable in a wide range of genetic investigations, including in situ analyses, genotyping and measurement of gene expression. However, padlock probes can be difficult to obtain by standard oligonucleotide synthesis because they are relatively long and require intact 5'- and 3'-end sequences to function. We describe a PCR-based protocol for flexible small-scale enzymatic synthesis of such probes. The protocol also offers the advantage over chemical synthesis that longer probes can be made that are densely labeled with detectable functions, resulting in an increased detection signal. The utility of probes synthesized according to this protocol is demonstrated for the analysis of single nucleotide variations in human genomic DNA both in situ and in solution.
Antson
D O
DO
Rudbeck Laboratory, Department of Genetics and Pathology, SE-751 85, Uppsala, Sweden and Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands.
Isaksson
A
A
Landegren
U
U
Nilsson
M
M
D000251:Adenosine Triphosphatases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D027682:Cation Transport Proteins; D002478:Cells, Cultured; D000073840:Copper-Transporting ATPases; D004252:DNA Mutational Analysis / Q000379:methods*; D004270:DNA, Circular / Q000096:biosynthesis / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D008677:Metaphase; D015345:Oligonucleotide Probes / Q000096:biosynthesis* / Q000138:chemical synthesis / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*
D002352:Carrier Proteins; D027682:Cation Transport Proteins; D004270:DNA, Circular; D015345:Oligonucleotide Probes; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases