Help:Primers/Problems using VF2 and VR to amplify BioBrick parts

Samantha Burke, as a UROP working at the Registry, identified a potential issue using VF2 and VR to amplify certain BioBrick parts via PCR. Her efforts are documented here.

Note: While VF2 and VR may yield extra bands during PCR of certain BioBrick parts as described below, both primers have been used successfully sequence a wide variety of BioBrick parts.

Figure 1: Extra bands are observed when two BioBrick parts are amplified by VF2 and VR. The extra bands are shorter and dimmer than the expected product. Columns 3-6 contain the template BBa_S03582, and columns 7-10 contain template BBa_I13033.

Primers VF2 and VR (Parts BBa_G00100 and G00101, respectively) are used to verify the length of parts in BioBrick plasmid backbones. When using them to PCR composite parts BBa_S03582 or BBa_I13033, I should observe a single band of 508 base pairs. Instead, I observed extra bands during colony PCR (Figure 1). To eliminate the additional bands, I performed a gradient PCR in which identical colony PCR samples were thermocycled using calculated annealing temperatures of 55.4°C, 56.7°C, 58.5°C, 60.9°C, 63.6°C, 65.8°C, 67.6°C, and 68.7°C. The extra bands are observed at every annealing temperature (Figure 2).

Upon inspection of the sequence of the two parts, I found that both part sequences contained regions that the primers could potentially bind and result in the shorter bands observed.

Click the gel images to view at larger size.

Figure 2: Gel electrophoresis results of gradient PCR of BBa_S03582 (top row) and BBa_I13033 (bottom row) using primers VF2 and VR. Samples are loaded in every other well. Annealing temperature increases from left to right.

Mispriming by VF2

Figure 3: Diagram of potential VF2 binding site in BBa_R0062.	Figure 4: Colony PCR of BBa_I13033, which contains BBa_R0062, using primers VF2 and VR.	Figure 5: Potential VF2 and VR mispriming sites in BBa_I13033.
VF2 can potentially bind BBa_R0062 (Figure 3). If VF2 anneals to BBa_R0062 as shown, a 220 base pair band would be produced by the PCR of BBa_I13033 with VF2 and VR. Although the potential primer binding site is not GC rich, there is an exact match of 5 nucleotides at the 3' end of the primer. The sequence of BBa_I13033 and all potential VF2 and VR mispriming sites is shown (Figure 5). Click the images to view at larger size.

Mispriming by VR

Figure 6: Diagram of potential VR binding site in BBa_B0010.	Figure 7: Colony PCR of BBa_S03582, which contains BBa_B0010, using primers VF2 and VR.	Figure 8: Potential VF2 and VR mispriming sites in BBa_S03582.
VR can potentially bind BBa_B0010 (Figure 6). The popular terminator BBa_B0015 contains BBa_B0010. The identified mispriming site shows that 10 of the 13 3' nucleotides of VR are complementary. If VR anneals to B0010 as shown, a 183 bp band would be produced by the PCR of BBa_S03582 with VF2 and VR. Colony PCR of BBa_S03582 using primers VF2 and VR show the expected band of 508 bp and the misprimed band of 183 bp (Figure 7). The sequence of BBa_S03582 and all potential VF2 and VR mispriming sites is shown (Figure 8). Click the images to view at larger size.