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

Published online by Cambridge University Press:  02 July 2020

Paul M. Lizardi ,
Patricia Bray-Ward  and
David C. Ward
Paul M. Lizardi
Affiliation:
Department of Genetics, Yale University School of Medicine, New Haven, CT, 06510
Patricia Bray-Ward
Affiliation:
Department of Genetics, Yale University School of Medicine, New Haven, CT, 06510
David C. Ward
Affiliation:
Department of Genetics, Yale University School of Medicine, New Haven, CT, 06510
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Extract

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.

Type
Genomes, Chromsomes and Genes: Emerging Technologies
Information
Microscopy and Microanalysis , Volume 3 , Issue S2: Proceedings: Microscopy & Microanalysis '97, Microscopy Society of America 55th Annual Meeting, Microbeam Analysis Society 31st Annual Meeting, Histochemical Society 48th Annual Meeting, Cleveland, Ohio, August 10-14, 1997 , August 1997 , pp. 201 - 202
Copyright
Copyright © Microscopy Society of America 1999

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References

1.Nilsson, M. et al., Science 26 (1994)2085.10.1126/science.7522346CrossRefGoogle Scholar
2.Heiskanen, M., et al., Genetic Analysis: Biomolecular Engineering 12 (1966)179.10.1016/S1050-3862(96)80004-0CrossRefGoogle Scholar