Phi29 dna polymerase reaction buffer

DNA extracts which had a qPCR CT value < 30 were tested using the Qubit for DNA concentration. Using this CT threshold, n = 145 samples were selected for further analysis. DNA concentration in the extracted dried blood spot samples was very low, of the range 0.011 ng/μl to 0.268 ng/μl. Selective whole genome amplification (SWGA) was used to amplify the Plasmodium DNA prior to sequencing, using a protocol adapted from Oyola et al.^{7 (link)}. Each SWGA reaction used 30 µl of extracted DNA, which was combined with 5 µl of phi29 DNA Polymerase Reaction Buffer (New England Biolabs), 0.5 µl of Recombinant Albumin (New England Biolabs), 0.5 µl of SWGA primers (250 mM) (Roche), 5 µl dNTPs (New England Biolabs) and 6 µl of water, to a total volume of 50 µl per reaction. SWGA reactions were then run in a thermocycler using the following programme steps: 35 °C for 5 min, 34 °C for 10 min, 33 °C for 15 min, 32 °C for 20 min, 31 °C for 25 min, 30 °C for 16 h, 65 °C for 10 min, and held at 4 °C. SWGA reactions increased the DNA concentration of samples to > 300 ng DNA in of the 48 of the 145 (33.1%) samples amplified.

Circular ligation of pooled (by mass) DNA was performed in 3 ml reaction volume with T4 DNA ligase (M0202, New England Biolabs, Ipswich, MA, USA) according to manufacturer’s protocol. The mixture was incubated at 16 °C for 6 h before heat inactivation of T4 DNA ligase at 65 °C for 10 min. Ligated products were purified and concentrated using AMPure XP beads. Plasmid-Safe ATP-Dependent DNase (E3101, Epicentre, Madison, WI, USA) digest was performed according to manufacturer’s protocol at 37 °C for 1 h to remove non-circularized products. DNase was then heat inactivated at 70 °C for 30 min. Products were purified and concentrated using AMPure XP beads. The circular DNA template molecules were added to phi29 DNA polymerase reaction buffer (B0269, New England Biolabs) with 0.25 μg/μl BSA, 5 μM exo-resistant random primers (S0181, Thermo Fisher Scientific), 0.25 mM dNTP (N0447, New England Biolabs) and 10 U phi29 polymerase (M0269, New England Biolabs) in a total volume of 20 μl. The conditions for RCA were set up as follows: 95 °C for 3 min, 30 °C (ramp rate of 0.1 °C/s) for 6 h and 65 °C for 10 min. The time taken for amplification was optimized to yield substantial RCA products, while minimizing the possibility of sequences produced by unspecific priming of random primers. RCA products were finally purified with 0.45× volume of AMPure XP beads.