Abstract
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.
Highlights
Cy3 Long circularization oligo for FITC and Cy5 Ligation template Detection oligo Cy3 Detection oligo FITC
Each copy of the rolling circle amplification product (RCP) contains parts used for hybridization to fluorophore-labeled detection oligonucleotides
As an attempt to reduce the size of the RCPs we designed a cross-hybridizing probe, which is composed of two identical sequences with each copy reverse-complementary to a part of each repeat in the RCP
Summary
Cy3 Long circularization oligo for FITC and Cy5 Ligation template Detection oligo Cy3 Detection oligo FITC. Is created by ligation of the juxtaposed two ends of the padlock probe. It has been thought that an RCP collapse onto itself by the polarity inherent to DNA, to fold into a random coil conformation[6]. An obstacle with RCA-based methods is that with an increased concentration of RCPs, the RCPs start to coalesce and individual products cannot be discerned[7]. It is desirable to develop RCA based assays that can generate signals smaller in size for an increased dynamic range. These properties facilitate a more accurate image analysis for RCA-based methods
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