Rolling Circle Amplification of Cyclizable Dna Probes by ∅29 Dna Polymerase: A Tool for in Situ Single-Copy Gene Detection.

1997 ◽  
Vol 3 (S2) ◽  
pp. 201-202
Author(s):  
Paul M. Lizardi ◽  
Patricia Bray-Ward ◽  
David C. Ward
Keyword(s):  
Dna Polymerase ◽  
Rolling Circle Amplification ◽  
Single Copy ◽  
Rolling Circle Replication ◽  
Gene Detection ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Cytological Localization ◽  
High Degree

Oligonucleotide probes that can be cyclized by ligation (“padlock probes”) provide a very high degree of recognition specificity. Nilsson et al. have demonstrated the used of padlock probes for the cytological localization of alphoid repeats in chromosome 12. We have been extending the use of padlock probes to the detection of single copy sequences, and with this in mind have explored the amplification of DNA circles.We designed a primer complementary to the arbitrary backbone (non-probing) sequence of a 92-base closed circular probe oligonucleotide and investigated the kinetics of rolling circle replication. Using the highly processive, strand-displacing DNA polymerase of phage ∅29 (kindly provided by Dr. Margarita Salas, CSIC, Madrid, Spain) we demonstrated that several hundred tandem copies of the circular oligonucleotide are generated in a few minutes of incubation at 32°C. Because the amplified DNA remains hybridized to the circle in a rolling circle reaction, this method of amplification offers unique advantages for in situ gene detection since the amplified DNA can not diffuse away from the site of synthesis.

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

2004 ◽  
Vol 1 (3) ◽  
pp. 227-232 ◽  
Author(s):  
Chatarina Larsson ◽  
Jørn Koch ◽  
Anders Nygren ◽  
George Janssen ◽  
Anton K Raap ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Dna Molecules ◽  
Rolling Circle ◽  
Padlock Probes

scholarly journals In vitro evolution of phi29 DNA polymerases through compartmentalized gene expression and rolling-circle replication

2019 ◽  
Vol 32 (11) ◽  
pp. 481-487 ◽  
Author(s):  
Yoshihiro Sakatani ◽  
Ryo Mizuuchi ◽  
Norikazu Ichihashi
Keyword(s):  
Gene Expression ◽  
Dna Polymerase ◽  
Multiple Displacement Amplification ◽  
Rolling Circle Amplification ◽  
Expression System ◽  
Rolling Circle Replication ◽  
Rolling Circle ◽  
Dna Labeling ◽  
Phi29 Dna Polymerase

Abstract Phi29 DNA polymerase is widely used for DNA amplification through rolling-circle replication or multiple displacement amplification. Here, we performed completely in vitro artificial evolution of phi29 DNA polymerase by combining the in vitro compartmentalization and the gene expression-coupled rolling-circle replication of a circular DNA encoding the polymerase. We conducted the experiments in six different conditions composed of three different levels of inhibitor concentrations with two different DNA labeling methods. One of the experiments was performed in our previous study and the other five experiments were newly conducted in this study. Under all conditions, we found several mutations that enhance the rolling-circle amplification by the polymerase when it was expressed in the reconstituted gene expression system. Especially, a combinatorial mutant polymerase (K555T/D570N) exhibits significantly higher rolling-circle activity than the wild type. These highly active mutant polymerases would be useful for various applications.

scholarly journals Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification

2011 ◽  
Vol 8 (1) ◽  
pp. 37 ◽  
Author(s):  
Sara Henriksson ◽  
Anne-Lie Blomström ◽  
Lisbeth Fuxler ◽  
Caroline Fossum ◽  
Mikael Berg ◽  
...  
Keyword(s):  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Replicative Form ◽  
Rolling Circle ◽  
Padlock Probes ◽  
In Situ Assay

Amplification of Padlock Probes for DNA Diagnostics by Cascade Rolling Circle Amplification or the Polymerase Chain Reaction

1999 ◽  
Vol 123 (12) ◽  
pp. 1170-1176 ◽  
Author(s):  
David C. Thomas ◽  
Glenn A. Nardone ◽  
Sandra K. Randall
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Polymerase ◽  
Rolling Circle Amplification ◽  
Strand Displacement ◽  
Chain Reaction ◽  
Dna Diagnostics ◽  
Gene Target ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Polymerase Chain

Abstract Context.—Padlock probes are highly specific reagents for DNA diagnostics that can discriminate gene sequences with single base mutations. When the 3′ and 5′ terminal regions of the oligonucleotide probes are juxtaposed on a target DNA sequence, they can be circularized by enzymatic ligation and become topologically locked to the target. However, to be useful in solution-based diagnostics, the sensitivity of padlock probes must be markedly enhanced. Objective.—To describe two methods for geometric amplification of circularized padlock probes. Design.—Cascade rolling circle amplification is an isothermal system that uses generic primers and a DNA polymerase with strong strand displacement activity to amplify circularized probes by a mechanism combining rolling circle replication and strand displacement synthesis. One of the primers was designed as an energy transfer–labeled primer, which generates a fluorescence signal only when incorporated into the amplified product, enabling a direct means of detection. Results.—Using pUC19 as a model target to circularize an 89-base probe, a 10 billion–fold amplification was achieved with Bst DNA polymerase (large fragment) within 1 hour starting with as few as 10 probe molecules. The polymerase chain reaction was also used to amplify ligated padlock probes in a rare target detection system. In mixing experiments containing both normal and mutant p53 or c-Ki-ras2 gene target sequences, mutant targets were easily detected in the presence of a 500-fold excess of normal target copies. Conclusion.—These results indicate that padlock probes can be amplified to the high levels required for solution-based DNA diagnostics.

scholarly journals Direct RNA targeted transcriptomic profiling in tissue using Hybridization-based RNA In Situ Sequencing (HybRISS)

2020 ◽  
Author(s):  
Hower Lee ◽  
Sergio Marco Salas ◽  
Daniel Gyllborg ◽  
Mats Nilsson
Keyword(s):  
De Novo ◽  
Detection Efficiency ◽  
Rolling Circle Amplification ◽  
Fold Increase ◽  
Data Interpretation ◽  
Rolling Circle ◽  
Padlock Probes ◽  
Multiple Transcripts ◽  
Detection Approach

Highly multiplexed spatial mapping of multiple transcripts within tissues allows for investigation of the transcriptomic and cellular diversity of mammalian organs previously unseen. Here we explore the possibilities of a direct RNA (dRNA) detection approach incorporating the use of padlock probes and rolling circle amplification in combination with hybridization-based in situ sequencing (HybISS) chemistry. We benchmark a dRNA targeting kit that circumvents the standard reverse transcription limiting, cDNA-based in situ sequencing (ISS). We found a five-fold increase in transcript detection efficiency when compared to cDNA-based ISS and also validated its multiplexing capability by targeting a curated panel of 50 genes from previous publications on mouse brain sections, leading to additional data interpretation such as de novo cell typing. With this increased efficiency, we maintain specificity, multiplexing capabilities and ease of implementation. Overall, the dRNA chemistry shows significant improvements in target detection efficiency, closing the gap between the gold standard of fluorescent in situ hybridization (FISH) based technologies and opens up possibilities to explore new biological questions previously not possible with cDNA-based ISS, nor with FISH.

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