Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

Abstract

Background: In situ detection of short sequence elements in genomic DNA requires short probes with high molecular resolution and powerful specific signal amplification. Padlock probes can differentiate single base variations. Ligated padlock probes can be amplified in situ by rolling circle DNA synthesis and detected by fluorescence microscopy, thus enhancing PRINS type reactions, where localized DNA synthesis reports on the position of hybridization targets, to potentially reveal the binding of single oligonucleotide-size probe molecules. Such a system has been presented for the detection of mitochondrial DNA in fixed cells, whereas attempts to apply rolling circle detection to metaphase chromosomes have previously failed, according to the literature.

Direct detection of RNA in vitro and in situ by target-primed RCA: The impact of E. coli RNase III on the detection efficiency of RNA sequences distanced far from the 3′-end

“…As a result, with the assistance of E. coli RNase III, the in situ detection of the inner RNA sequences located either near the end or in the middle of the RNA molecule (hGAPDH) became comparably efficient (4% and 7%, respectively). Similar detection efficiencies in situ (1%-10%) were presented previously for the DNA-primed RCA technique on the mitochondrial or chromosomal DNA targets (Larsson et al 2004;Lohmann et al 2007). …”

Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA

“…Therefore obtained detection efficiency of target GAPDH transcript-primed RCA in situ was z1% relative to the RT-qPCR outcome. Similar to target DNAprimed RCA (overall detection efficiency of the mitochondrial or chromosomal DNA targets' in situ was 1%-10%) (Larsson et al 2004;Lohmann et al 2007) current detection efficiency of the target RNA-primed RCA technique in situ precludes the application of this method for single-copy target examination in single cell. However, it still can be used for the detection and analysis of high-copy RNA transcripts as shown for GAPDH transcript and needs additional improvements and efforts rendering the method suitable for lowcopy RNA transcript analysis.…”

Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly

“…Padlock probes were introduced about a decade ago to detect single base variations in FISH format (Christian et al, 2001; Larsson et al, 2004; Lohmann et al, 2007). This technique is based on the extremely high sequence specificity of the ligation reaction that can discriminate single mutations if they are located close to the ligation point.…”

“…This method can be used to detect individual DNA molecules with great specificity, but the efficiency of the detection is about 10%. Lohmann et al attempted to employ target-primed RCA for detection of DNA on metaphase chromosomes (Lohmann et al, 2007). However, this method was only able to detect 1–10% of the target sites, and the authors decided that this technique is best suited for the targets that exist in multiple copies.…”