Fig 1.
Schematics of the subcycling PCR protocol.
A. Each PCR cycle involves a denaturation step and an annealing/elongation step. We introduced 4X sub-cycling the annealing/elongation step within each of the 30X amplification cycles. B. Multiplexed amplification products for pools of 7 oligos with 154–200 bp length products of varying GC content. Phusion and KAPA HIFI polymerases were used with and without a sub-cycling thermocycle. Each different condition is used to amplify 12 separate oligo pools with GC content ranges as follows: 1.) 16.4–34.3; 2.) 13.5–38.7; 3.) 21.5–37.3; 4.) 12.2–12.2; 5.) 12.7–40.0; 6.) 14.9–41.6; 7.) 16.4–37.6; 8.) 12.7–42.0; 9.) 20.9–40.6; 10.) 12.5–42.5; 11.) 12.7–43.7; 12.) 14.8–35.6. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 154-200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.
Fig 2.
Oligonucleotides copy numbers following multiplex PCR amplification correlated with their GC content.
A. Standard PCR amplification; B. Subcycling PCR amplification. Note: All oligonucleotides with matching colors and shapes were amplified in the same pool in a multiplexed PCR reaction. Pools were then subjected to a MiSeq analysis and each data point represents the copy number of one oligonucleotide.
Fig 3.
PCR amplification of high and low GC oligonucleotides with and without subcycling and varying additives.
A. 10% GC, no sub-cycling. B. 79% GC, no sub-cycling. C. 10% GC, sub-cycling. D. 79% GC, sub-cycling. First lane on the left of each gel: MW markers. Lanes numbered as follows: 1) no additive 2) 40:60 dGTP:deaza-dGTP 3) 50:50 dGTP:deaza-dGTP 4) 60:40 dGTP:deaza-dGTP 5) 2.5% DMSO 6) 10% DMSO 7) 0.1M betaine 8) 0.2M betaine 9) 0.4M betaine 10) 50:50 dGTP:deaza-dGTP with 5% DMSO 11) 60:40 dGTP:deaza-dGTP with 10% DMSO. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows an example of an expected PCR pattern where a strong 200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.
Fig 4.
PCR amplification of oligonucleotides with wide range GC contents with subcycling and different additives.
A. No additives; B. 60% deaza-dGTP; C. 0.2M betaine. First lane on the left of each gel: MW markers. Lanes numbered indicates oligonucleotides of varying GC content as follows: 1) 10% GC; 2) 21%GC; 3) 33%GC; 4) 44%GC; 5) 56%GC; 6) 67%GC; 7) 79% GC; 8) 90%GC. Results are based on electronic gels created by electrophoresis using a Perkin Elmer GX instrument with a 5k chip. *Bin shows expected PCR pattern where a strong 200bp product band is seen. PCR reactions were not purified and primers can be seen at the bottom of each sample.
Fig 5.
Percent of successful builds of DNA constructs with varying GC content.
The bar graph is a visual representation of the data in Tables 1 and 2. Blue bars—represent the successful builds with the standard protocol. Red bars—represent the successful builds with the broad spectrum protocol.
Table 1.
Percent of successful builds of DNA constructs with varying GC content using the standard protocol.
The same data is visualized as a bar graph in Fig 5.
Table 2.
Percent of successful builds of DNA constructs with varying GC content using the broad spectrum protocol.
The same data is visualized as a bar graph in Fig 5.