Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells

Abstract

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays. R olling circle amplification (RCA) is a molecular cytogenetic technique used with a padlock oligonucleotide probe to detect single base changes in isolated nucleic acids (1-5). Padlock probes are composed of Ϸ100 nucleotides that hybridize to targets of Ϸ30 bases. The 30-base target-binding region of the probe is split into two 15-base segments placed in opposite orientation at each end of the linear probe so that a circle must be formed for hybridization to occur (6, 7). At 10 bases per helical turn, the hybridized probe wraps around its target three times, and the remaining 70 bases form an unhybridized singlestranded loop. Posthybridization DNA ligation connects the two ends of the probe in the middle of the 30-base binding region. The unbound 70-base loop facilitates probe circularization and permits Ϸ20 bases to serve as a primer recognition site for DNA polymerase to replicate the circle. RCA is an isothermal process in which the polymerase progresses continuously around the loop until the 100 bases have been replicated hundreds or thousands of times. Incorporating a labeled nucleotide during the RCA reaction produces sufficient signal for easy visualization of the target.Application of RCA to in situ targets in fixed or permeabilized cells has not been uniformly successful to date. Whereas recent work has demonstrated that the concept is viable (8), DNA detection efficiencies of 20-30% lessen the utility of RCA as an assay. Lack of success has been attributed to possible blocking of the polymerase by the target strand, and it was suggested that this problem might be overcome by cutting the target DNA strand near the RCA probe's hybridization site (5). Under these conditions, DNA polymerase could free the probe from the target, in effect spinning the probe away from the target, keeping the polymerase from being blocked during the amplification process. Here, we report that in addition to restriction enzyme digestion of DNA, additional steps were required to achieve consistent and satisfactory results for RCA in situ. Whereas heat denaturation is typically used to render the target DNA single stranded, we found that complete removal of the nontargeted DNA strand by digestion with exonuclease III significantly increased the efficiency of the process.We also demonstrate the use of RCA to detect mRNA in cytological preparations. Using appropriate image analysis techniques, the RCA assay is sufficiently quantitative to enable transcriptionally mediated dose-response curves to be generated. Inc...

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

“…We modified an enzymatic target preparation procedure previously used for in situ amplification of padlock probes 15 to produce single-stranded target strands with free 3¢ ends downstream of the recognition sequences for the padlock probes, allowing target-primed amplification of circularized probes. Thus, the target strand can first act as the template for circularization of the probes and then prime RCA, thus avoiding topological inhibition of replication and ensuring efficient retention of the amplification products at the original location of the target sequences (Fig.…”

“…We carried out experiments to compare the present method to previously published in situ RCA procedures 12,15 . The first strategy used preformed circular DNA strands to template RCA reactions in situ to detect the hybridization of sequencespecific linear probes to their target sequences 12 .…”

“…Next, we compared our procedure with a strategy where target sequences with nearby 5¢ ends were obtained by enzyme treatment and padlock probes were amplified using an added oligonucleotide primer 15 . In this comparison we added the ppMUTs probe to samples where restriction digested target sequences had been made single stranded using exonuclease III, producing a free 5¢ end upstream of the probe recognition site.…”

“…If, instead, the reaction is set up so that the RCA template can form only in a target-specific circularization reaction in situ, nonspecific signals should be greatly diminished; and promising results have been reported with this approach 14,15 . Regrettably, replication of circular probes that remain wound around their target strands seems to be inhibited, probably as a consequence of molecular crowding 16 , although there is some controversy in the literature about this topological inhibition 17 .…”

“…Regrettably, replication of circular probes that remain wound around their target strands seems to be inhibited, probably as a consequence of molecular crowding 16 , although there is some controversy in the literature about this topological inhibition 17 . In an interesting approach, target sequences were enzymatically rendered single stranded having free 5¢ ends near the binding sites for padlock probes 15 . Upon addition of a primer the probes can be replicated in situ by RCA, but only if they escape the topological block against replication by detaching from the target molecules.…”