Hifi buffer

The cfDNA concentrations were determined as described by Neuberger et al. [22 (link)]. Ahead of the measurements, the linearity, limit of quantification, and limit of detection of the assay were determined. The qPCR assay amplifies DNA in unpurified plasma, which is diluted 1:10 in UltraPure DNase/RNase-Free H2O (Invitrogen, Waltham, MA), targeting a 90 bp fragment of human long interspersing nuclear elements (LINEs) of the repetitive L1PA2 family (5′-TGCCGCAATAAACATACGTG-3′ and 5′-GACCCAGCCATCCCATTAC-3′). Briefly, each sample was measured as a technical triplicate with 5 µL final volume containing 0.66 µL of 1:10 diluted plasma, 0.33 µL primer mix (140 nm final concentration of each primer) and 4 µL of qPCR mix with 0.6 U Velocity Polymerase (Bioline, London, UK), 1.2 × Hifi Buffer (Bioline, London, UK), 0.1 × SYBR Green (Sigma, St. Louis, MO, USA), and 0.3 mM dNTPs (Bioline, London, UK). The qPCR reaction was carried out using a CFX384 Bio-Rad (Bio-Rad, Munich, Germany) cycler with a two-step protocol. The cycling conditions were: initial heat activation at 98 °C for 2 min followed by 33 cycles of 95 °C for 10 s and 64 °C for 10 s with a subsequent melting curve from 70 to 95 °C with 0.5 °C increments for 10 s. The operator measuring the samples was blinded and did not know the allocation of the samples into the control or intervention group.

The hrp gene was cloned into the pET21a vector using NdeI/SalI restriction sites to generate the pET21a-HRP construct. The template DNA for CFPS was prepared by PCR using a forward primer for the 15 base pairs upstream of the T7 promoter (T7Pro-15UP), and a reverse primer for the T7 terminator (T7Ter). The PCR mixture consisted of 1 ng/ µl template, 200 nM of each (forward and reverse) primer, 0.02 U/µl DNA polymerase, 1 mM each of dNTP, and 1 × Hi-Fi buffer (Bioline). Thermal cycling involved heating at 95°C for 2 min, followed by 28 cycles at 95°C for 30 s, 55°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 2 min. PCR products were purified using a PCR Clean-up kit (Promega, Madison, WI, United States) prior to use for CFPS. For randomization of the +2 and +3 codons, the hrp gene was amplified using a forward primer containing degenerated +2 and +3 codons (+2/+3MutHRP, Supplementary Table S1), re-cloned into the pET21a plasmid, and transformed into competent E. coli DH5α cells. After overnight culturing of the transformed E. coli DH5α cells on Luria-Bertani (LB) agar plates, individual colonies were picked for colony PCR to prepare template DNA for CFPS.