Difference between revisions of "Problems with PCR using VR/VF2"

Revision as of 20:57, 22 August 2007

Results of a Colony PCR - the excess bands are shorter and dimmer than the expected product. Columns 3-6 is S03582, columns 7-10 is I13033.

VR and VF2 (Parts G00101 and G00100 respectively) are used to verify the lengths of parts. While using them to PCR potential parts (S03582 and I13033, both composite parts) for the Registry's Assembly Ladder, I noticed that I was consistently getting excess bands. Contamination was ruled out since the bands still appeared in a Colony PCR. Upon inspecting the sequence of the two parts, I found that each contained regions that the primers could bind, resulting in the shorter bands I was consistently seeing. Since S03582 and I13033 are both composite parts, I determined where exactly the primers were binding to see what parts would be affected during PCR with VR and VF2.

Sequence and Features for I13033

Sequence and Features for S03582

VR Incorrect Annealing

Diagram of potential VR binding sites within B0010

VR can potentially bind B0010 in three different ways. One of the annealing schemes, shown in red in the diagram and below, seems particularly likely. Only 3 of the last 13 bases of VR mismatch to B0010. Also, VR's 3' end would anneal the the part tightly due to the two G - C pairs. The other two possibilities, shown in blue and green, are less likely to occur during PCR. Although the primer may anneal to B0010 in one of the two ways, the 3' end of VR does not match closely enough in either case for extension to occur.

Colony PCR for S03582

For both S03582 and I13033, PCR with VR and VF2 should give a 508 base pair product. If VR anneals incorrectly in the manner depicted above the product would be 183 base pairs long. The close-up of the gel shows these two bands for S03582.

VF2 Incorrect Annealing

Diagram of potential VF2 binding sites within R0062

VF2 can potentially bind R0062 in two different places. Annealing to the segment shown in blue in the diagram seems unlikely to result in extension since there is not any significant pairing near the 3' end of the primer. Depending on the annealing and extension temperatures being used, VF2 may prime from the segment of R0062 shown in red. The binding would not be strong, since the matched bases are mostly A's and T's, but it could be enough to produce the excess bands in the PCR.

Colony PCR for I13033

If VF2 anneals to R0062 as shown above, a 220 base pair band would be produced by the PCR with VR and VF2.

Binding Diagrams for I13033 and S03582

Diagram for I13033

Diagram for S03582