Phi29 buffer

The ligation and amplification procedure for producing a stock solution containing 0.5 pmol (in 100 µL) RCA products has been reported earlier [23 (link)]. Briefly, a ligation mixture containing 3 pmol DNA target, 1 pmol phosphorylated padlock probe (biomers.net), 1 mM ATP (Thermo Fisher Scientific, Stockholm, Sweden), 20 mU/µL T4 ligase (Thermo Fisher Scientific), and 1 × phi29 buffer (Thermo Fisher Scientific) was incubated for 15 min at 37 °C in order to let the padlock probe hybridize to the target and form a molecule circle through ligation. Thereafter, the ligation mixture consisting of 1.2 pmol of circles was mixed with 167 µM dNTP (Thermo Fisher Scientific), 0.2 µg/µL bovine serum albumin (BSA; Thermo Fisher Scientific), 2.7 mU/µL phi29 DNA polymerase, and 1 × phi29 buffer, and the reaction was incubated at 37 °C for 60 min followed by enzymatic inactivation at 65 °C for 5 min. Hybridization buffer (0.1 M Tris-HCl (pH 8), 0.1 M EDTA, 0.5% Tween-20, 2.5 M NaCl) was then added to the RCA mix to obtain the desired RCA product concentration. Duplicates samples of 0.5 pmol RCA products were made. Samples containing 1, 10, and 100 fmol of RCA products were prepared through mixing appropriate amounts of the RCA samples with hybridization buffer. Water was used instead of DNA target in the ligation mixture for the negative control (NC) samples.

Padlock oligonucleotides, including a 15 nt random sequence (100 nM) were ligated into circular DNA strands in the presence of template (100 nM) in 1x phi29 buffer ((Thermo Scientific), 10 mM Mg-acetate, 66 mM K-acetate, 0.1% (v/v) Tween 20, 1 mM DTT) containing 0.01 U/μl T4 ligase (Thermo Scientific), and 1 mM ATP (Thermo Scientific). The ligation was performed at 37 °C for 30 min. To remove unligated padlock oligonucleotides, exonuclease I and exonuclease III (Thermo Scientific) were added to the ligation mix to a concentration of 0.2 U/μl and 2 U/μl, respectively. The reactions were incubated at 37 °C for 30 min and terminated by incubation at 85 °C for 20 min. Then the RCA primer (the same oligonucleotide used as ligation template) was adjusted to a concentration of 100 nM and incubated at 37 °C for 20 min to reanneal to the circular DNA strands. The RCA reactions were initiated by adding d(A, U, G, C)TP at concentrations of 1 mM and phi29 polymerase (Thermo Scientific) at 0.1 U/μl. RCA was performed at 37 °C for 10 min, and terminated by heating at 65 °C for 10 min. The RCA products were kept at −20 °C until use. The RCA products were characterized with Nanoparticle Tracking Analysis NTA (Malvern Nanosight NS300) according to the manufacturer’s instructions.

Phi29 polymerase, phi29 buffer, dNTP mix, bovine serum albumin (BSA), GeneRuler low range DNA ladder, SYBR Gold nucleic acid gel stain, Tris-HCl buffer (1 M, pH 8.0) and Tris-acetate-EDTA buffer (TAE, 50 ×) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CircLigase II ssDNA ligase, together with other ligation reagents (buffer, MnCl₂, betaine), was purchased from Biosearch Technologies (Novato, CA, USA). Nb.BtsI nickase, thermolabile exonuclease I, exonuclease III and loading buffer were purchased from New England BioLabs (Ipswich, MA, USA). Recombinant Acidaminococcus sp. BV3L6 Cas12a nuclease was purchased from Integrated DNA Technologies (Coralville, IA, USA). Fetal bovine serum (FBS), salmon sperm DNA and agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). Streptavidin-coated cross-linked starch iron oxide composite particles (100 nm size MNP) were purchased from Micromod Partikeltechnologie GmbH (Rostock, Germany). DNA and RNA sequences were synthesized by Integrated DNA Technologies and diluted in 50 mM Tris-HCl (pH 8.0). Sequences of targets (target dengue sequence and target B for amplifying detection loop and reference loop, respectively), linear templates (linear detection template and linear reference template), crRNA and detection probes (DP-DL-I, DP-DL-II, DP-RL-I and DP-RL-II) are listed in Supplementary Table S1.