Rolling Circle Amplification (RCA) by Vadim V. Demidov

Author:Vadim V. Demidov
Language: eng
Format: epub
Publisher: Springer International Publishing, Cham

2.2 RCA on Glass Slides and Visualization of Amplicons

A 5′-phosphorylated 88-nt-long oligonucleotide was converted into a circular DNA probe by T4 DNA ligase in the presence of biotinylated complementary “splint” oligonucleotide, also used as a primer in RCA on slide. The aliquot of circular DNA probe and primer mixture (10 μL) was added to commercially available streptavidin-coated glass slides (105 streptavidin molecules per mm2). After a few minutes to allow binding of all components to the slide (see Fig. 8.1a), 40 μL of solution containing RCA reaction mix (1 U of Phi29 DNA polymerase and 20 mM dNTPs in the buffer containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2, 25 mM NaCl, and 5 mM DTT) was added. The components of the mixture were incubated at 37 °C for 4 h. Note that 10 % of dCTP was substituted by the Cy3-fluorescent dye to permit visualization of the rolling circle product (amplicon). The region containing the reaction mix was covered and sealed with adhesives. Then the samples were washed by immersing the slides in (a) 2× SSC with 2 % SDS, (b) 2× SSC with 50 % formamide, (c) 2× SSC, and (d) 0.2× SSC. The slide was air-dried and subjected to GenePix fluorescence-imaging scanner analysis. The images were processed by ImageJ software.

Fig. 8.1(a) Schematic of RCA strategy on slides. (b, c) Detection of individual RCA amplicons on glass slide coated with (b) 102/sq. mm molecules and (c) 101/sq. mm molecules of DNA minicircle bound to DNA tag primer. Reproduced with permission (Konry et al. 2011). Copyright Wiley-VCH Verlag GmbH & Co. KGaA

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