Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio

Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate t...

Full description

Saved in:
Bibliographic Details
Published in:Scientific reports 2015-07, Vol.5 (1), p.12317-12317, Article 12317
Main Authors: Clausson, Carl-Magnus, Arngården, Linda, Ishaq, Omer, Klaesson, Axel, Kühnemund, Malte, Grannas, Karin, Koos, Björn, Qian, Xiaoyan, Ranefall, Petter, Krzywkowski, Tomasz, Brismar, Hjalmar, Nilsson, Mats, Wählby, Carolina, Söderberg, Ola
Format: Article
Language:English
Subjects:
13/51
14/10
631/1647/2234
631/1647/245/2225
631/61/32
Compaction
Computerized Image Processing
Datoriserad bildbehandling
Deoxyribonucleic acid
DNA
DNA probes
DNA, Circular - chemistry
DNA, Circular - genetics
DNA, Single-Stranded - chemistry
DNA, Single-Stranded - genetics
Fluorophores
Humanities and Social Sciences
Hybridization
multidisciplinary
Nucleic Acid Amplification Techniques - methods
Oligonucleotides
Phospholipase A
Reproducibility of Results
Science
Sensitivity and Specificity
Sequence Analysis, DNA - methods
Signal-To-Noise Ratio
Single-stranded DNA
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep12317