Detection efficiency of bead-based rolling circle amplification (RCA)

Range ~1.3 %
Organism Bacteria Salmonella enterica
Reference Sasaki N, Gunji Y, Kase C, Sato K. Molecular crowding improves bead-based padlock rolling circle amplification. Anal Biochem. 2016 Dec 7 519: 15-18. doi: 10.1016/j.ab.2016.12.002. p.15 rightt column top paragraphPubMed ID27940012
Primary Source [3] K. Sato, R. Ishii, N. Sasaki, K. Sato, M. Nilsson Bead-based padlock rolling circle amplification for single DNA molecule counting Anal. Biochem., 437 (2013), pp. 43–45 doi: 10.1016/j.ab.2013.02.016.PubMed ID23467098
Comments P.15 left column bottom paragraph to right column top paragraph: "Bead-based padlock RCA is a technique [investigators] developed to improve detection efficiency [ref 2]. The principle of the technique is shown in Fig. 1A. A padlock probe is circularized in the presence of sample DNA and T4 DNA ligase. The circularized probe is hybridized with primer DNA anchored to the microbead. The primer is extended along the circularized probe by phi29 DNA polymerase, which possesses strong strand displacement activity, and long single-stranded DNA is produced. The RCA products are anchored to the beads, which simplifies observation of the products. As a result, quantitative detection of Salmonella DNA was achieved, with a limit of detection of 9 pM [primary source]. The detection efficiency in bead-based RCA is estimated to be ∼1.3% (1 fmol sample DNA, 1.0×10^4 beads, 778 products per bead) [primary source]. This suggests that, even in bead-based RCA, not all of the samples react with the padlock probes, or that not all of the circularized probe is used to extend the primers (Fig. 1B). Therefore, in an effort to improve the detection efficiency, a more fundamental solution is required." See BNID 113083
Entered by Uri M
ID 113084