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

Abstract

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, oligo-nucleotides 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.

The peptide nucleic acids (PNAs), powerful tools for molecular genetics and cytogenetics

“…43,44 Chen et al 45 reported that PNA probes could discriminate in situ between two centromeric DNA repeats that differ by only a single base pair. Similar results were obtained with PRINS primers, oligonucleotide probes or padlock probes, 32,46,47 but never with standard FISH probes. The identification of chromosomal variations and the analysis of polymorphisms could greatly benefit from the discrimination power of PNAs.…”

Hydrogen peroxide scavenging rescues frataxin deficiency in a Drosophila model of Friedreich's ataxia

“…A role for frataxin in preventing formation of deleterious reactive oxygen species (ROS) has been well established (12,15,24), invoking a paradigm of FRDA pathology in which ROS toxicity leads to mitochondrial dysfunction with subsequent cell death (for review, see refs. 25 and 26).…”

In situ genotyping individual DNA molecules by target-primed rolling-circle amplification of padlock probes

“…These probes have detection specificity similar to that obtained with PCR [1][2][3] , but unlike in PCR, the reaction products can remain bound to their target sequences, even withstanding denaturing washes 4 . Unfortunately, detection signals from specifically circularized padlock probes have proven insufficient to reveal single-copy gene sequences owing to background noise from nonspecifically bound probes, although repeated centromeric sequences have been evaluated with sufficient precision to investigate the distribution of single-nucleotide variants in situ 5,6 .…”