Padlock probes reveal single-nucleotide differences, parent of origin and in situ distribution of centromeric sequences in human chromosomes 13 and 21

Chromosome centromeres, composed of repeated DNA sequences, orchestrate the correct segregation of chromatids in cell division. We have examined the centromeres of human chromosomes 13 and 21 by studying the distribution, in situ, of two alpha satellite sequences that differ in a single nucleotide position. This was possible using padlock probes, oligonucleotides that can be ligated into circles upon target recognition. The segregation of individual 13 and 21 homologues in a family was followed by monitoring of the signals from two differentially labelled probes, specific for either sequence variant. A characteristic arrangement of the repeat motifs in three separate spots-oriented transverse to the length axis of the metaphase chromosomes and bilaterally symmetric, indicates that only parts of the detected regions are involved in the centromeric region, joining the sister chromatids before anaphase.