Strategy for accurate sequencing through hairpin structures

Verifying the sequence of an shRNA hairpin is essential
since mismatch of even one nucleotide within the target sequence can ablate knockdown . An issue that is frequently encountered in the preparation of shRNA vectors is that many are difficult to sequence due to the intrinsic secondary structure of the hairpin. One strategy recently proposed to overcome this issue involves engineering a restriction site within the loop/stem region of the hairpin to physically separate the inverted repeats by digestion, and then piecing together sequence using sense and antisense primers . However, the ability to achieve sequencing of shRNA constructs without modifying
stem/loop sequence would be of clear advantage. To address this possibility, we evaluated modified sequencing reactions for improvement in the read-through of the
hairpin secondary structure in three shRNA hairpins. Modifications include adding agents known to relax DNA structure including DMSO, Betaine, PCRx Enhancer and ThermoFidelase I; and adding increasing amounts of dGTP BigDye terminator (dGTP) chemistry to the standard BigDye v1.1 (BD) chemistry which contains dITP rather than dGTP.

Sequencing results for each of the three DNA constructs are summarized in Table 2. Read-through of the hairpin structure was measured as the ratio of the peak height
about 300 bases after the hairpin structure to the signal about 50 bases before the hairpin structure. A ratio of 1 indicates no loss in signal and 0 indicates complete loss of
read-though. In the absence of any additive to BD chemistry, the hairpin caused a reduction in peak height ratio for our less tightly structured hairpin, pHSPG-shmutTLR4,
to 0.4, and a complete loss in read through for the other two plasmids. This can be visualized as an abrupt stop in the sequence peak profile for pHSPG-shTLR4 (Figure 3A).
Among the DNA relaxing agents, 5% DMSO, 0.83 M Betaine and 1 × PCRx Enhancer each improved the sequence read significantly for some constructs. However,the addition of 0.83 M Betaine plus 1 × PCRx Enhancer to BD chemistry was found to sequence most consistently, with peak height ratios of 0.5–0.9 .
The addition of 10:1 BD:dGTP chemistries alone also improved read through somewhat, with peak height ratios of 0.5–0.6 . The sub-optimal peak height ratio for 10:1 BD:dGTP can be attributed to a visible step in the sequence peak profile after the secondary structure region where the signal is reduced (Figure 3C,arrow). Increasing the dGTP chemistry content to 5:1 and 3:1 BD:dGTP or using straight dGTP chemistry increased the peak height ratio and reduced the step somewhat (0.6 to 0.8 ratio). However, the mixed incorporation of dITP and dGTP resulted in worse peak broadening as the
amount of dGTP used increased [see Additional file 1], and dGTP only chemistry caused severe sequence compressions (data not shown). The best overall results were
observed by combining Betaine plus PCRx and 10:1 BD:dGTP mixed chemistries together. This combination reduced the step with less peak broadening and increased
peak height ratios to 0.9–1.0 (Table 2 and Figure 3D).ThermoFidelase I, a DNA destabililizing enzyme that is frequently used to improve sequencing of genomic DNA
[25,26], did not improve sequencing of any of the three hairpins in straight BD chemistry (data not shown), and actually reduced the peak height ratio significantly in 10:1
BD:dGTP chemistries for all three shRNA constructs, causing the reappearance of a stop at the hairpin structure .
In summary, the combination of 10:1 BD:GTP chemistries,0.83 M Betaine, and 1 × PCRx Enhancer provided optimal sequencing, and mixed BD:dGTP chemistries,
Betaine, PCRx Enhancer, and DMSO each had some positive effects on their own. ThermoFidelase I, however,probably should be avoided for shRNA vectors with difficult
intrinsic secondary structure.