Expanded genetic alphabets in the polymerase chain reaction

Abstract

Cleaning up polymerase chain reactions: Artificially expanded genetic information systems (AEGIS) add extra nucleotide "letters" to DNA alphabets; oligonucleotides containing AEGIS nucleotides do not bind to natural DNA. This "orthogonality" is exploited here by placing two AEGIS nucleotides (P and Z) in external tags for primers targeting three cancer genes in a nested PCR architecture. AEGIS tags support multiplexed PCR with fewer primer dimers and off-target amplicons than multiplexed PCR without AEGIS components.

Figure 1

Comparison of the standard C:G pair with the non-standard Z:P pair forming an extra two genetic letters in an artificially expanded genetic information system (AEGIS).

Figure 2

Nested PCR with standard and AEGIS components using Taq DNA polymerase at four annealing temperatures (50.4, 54.2, 58.9, and 63.9 °C). The initial concentration of template DNA is 0.25 nM. a) Nested PCR with different combinations of external and composite primers. Type A nested PCR: Target first amplified by standard composite primers at low concentrations, and then standard external primers (A-Std) or AEGIS external primers (A-AEGIS) at high concentrations. Type B nested PCR: Target first amplified by AEGIS composite primers at low concentrations and then standard (B-Std) or AEGIS external primers (B-AEGIS). b) Autoradiograph of a PAGE gel resolving nested PCR products. Lanes 1 to 4 (A-Std): Products from composite primers and external primers both without AEGIS. Lanes 5 to 8 (A-AEGIS): Absence of products with standard composite primers and AEGIS external primers. Lanes 9 to 12 (B-Std): Absence of products with AEGIS composite primers and standard external primers. Lanes 13 to 16 (B-AEGIS). Products from composite and external primers both with AEGIS.

Figure 2

Nested PCR with standard and AEGIS components using Taq DNA polymerase at four annealing temperatures (50.4, 54.2, 58.9, and 63.9 °C). The initial concentration of template DNA is 0.25 nM. a) Nested PCR with different combinations of external and composite primers. Type A nested PCR: Target first amplified by standard composite primers at low concentrations, and then standard external primers (A-Std) or AEGIS external primers (A-AEGIS) at high concentrations. Type B nested PCR: Target first amplified by AEGIS composite primers at low concentrations and then standard (B-Std) or AEGIS external primers (B-AEGIS). b) Autoradiograph of a PAGE gel resolving nested PCR products. Lanes 1 to 4 (A-Std): Products from composite primers and external primers both without AEGIS. Lanes 5 to 8 (A-AEGIS): Absence of products with standard composite primers and AEGIS external primers. Lanes 9 to 12 (B-Std): Absence of products with AEGIS composite primers and standard external primers. Lanes 13 to 16 (B-AEGIS). Products from composite and external primers both with AEGIS.

Figure 3

Products from nested PCR amplification of Taq gene using standard or AEGIS-containing primers. Lane 1: Hi-LO™ DNA marker 50bp – 10,000pb (Bionexus). Lane 2: Control without target; composite and external primers without AEGIS. Lane 3: Standard nested PCR with standard primers. Lane 4: Control lacking composite primers, but with standard external primers and target. Lane 5: Control without target; composite and external primers with AEGIS. Lane 6: Standard nested PCR with AEGIS primers. Lane 7: Control lacking composite primers, but with AEGIS external primers and target.

Figure 4

Seven parallel nested PCR amplifications of three genes (TOP1, HBEGF, and MYC) from human genomic DNA (Phusion high fidelity DNA polymerase) under seven different annealing temperatures (53.4, 54.6, 56.5, 58.8, 61.5, 63.8 and 65.5 °C). Left: Amplification with standard external and composite primers. Right: Amplification using P-containing external and composite primers. Controls A (for standard nested PCR) and B (for P-containing nested PCR) were performed at 58.8°C without genomic DNA.