PADLOCK PROBES FURNISH NEW TOOLS FOR HIGH-CAPACITY GENETIC ANALYSES

Department of Genetics and Pathology, Box 589 BMC, Se-751 23 Uppsala, Sweden

Increasingly, molecular genetic investigations require analysis of very large sets of gene sequences in individual samples. Padlock probes offer a means to circumvent limitations of current gene detection methods (Nilsson et al. Science, 1994), 265: 2085). These reagents are linear oligonucleotides, with target-complementary sequences located at both ends, and separated by a spacer segment. Upon hybridization to a target-sequence, the two ends of the probes are brought in juxtaposition, and they can then be joined through enzymatic ligation. The circularization reaction is highly specific because of the requirement for hybridization by two probe-segments, and this intramolecular reaction is much less susceptible to problems of multiplexing than is PCR. Further, ligation easily discriminates among similar sequence variants. We have used the probes to monitor in situ the segregation of single-nucleotide variants of a centromeric repeated sequence in humans (Nilsson et al. Nature Genet, 1997, 16: 252). In order to extend the analysis to any single-copy sequence in complex genomes, conditions were defined under which circularized reacted probes can template a rolling circle mechanism, offering a powerful local signal amplification (Banér et al. Nucl Acids Res (1998) in press). We now apply these reagents in two important areas: 1) Circularizable reagents are immobilized as oligonucleotide arrays, suitable to detect, distinguish, and quantitate very large sets of gene sequnces in a sample, with no need for prior DNA amplification. 2) By applying similar probes in situ , it should be possible to type sets of allelic sequence variants as they occur along each homolog of a chromosome or a chromatin fiber, thus directly establishing haplotype information.