Background and Experiment
Multiplexing PCR amplification of full-length antibody coding sequences has been challenging in the past because high levels of primer dimers are formed. Crissman et al.  immunized mice and isolated B cells in 96-well plates. After a freeze-thaw cycle, they performed cDNA synthesis to obtain templates for PCR amplification of coding sequences for antibody variable regions. Two PCRs were performed; the nested second PCR included either standard primers or rhPCR primers and RNase H2 enzyme from IDT. The PCR products were cloned into plasmids which were used to transfect Chinese hamster ovary (CHO) cells. The CHO cells then produced the encoded antibodies and excreted the antibodies into the cell culture supernatant. Western blotting and ELISA were performed to detect the antibodies. In addition to the 96-well PCRs, Crissman et al. also performed 384-well PCRs to prepare samples for NGS. IDT primers were also used for these PCRs.
The PCRs were designed to amplify antibody sequences such as variable heavy (VH) and variable light (VL) chains. When PCR was performed using standard primers, amplicons were analyzed by agarose gel electrophoresis, and some of the VH samples showed that a small, unwanted PCR product of <150 bases had been amplified. After translation in CHO cells, these short products appeared on Western blot, demonstrating that they would be analyzed falsely as positives. Crissman et al. attempted to decrease the formation of this unwanted PCR product by a variety of approaches such as altering the temperatures used for primer annealing, decreasing the primer concentrations, changing the ratios of the forward and reverse primers, and decreasing the number of PCR cycles. None of these approaches made a difference to the amount of unwanted short PCR product synthesized.
After these unsuccessful attempts to decrease the unwanted small PCR products resulting from use of standard primers, Crissman et al. used two varieties of primers from IDT. These were the Generation 1 and Generation 2 rhPCR primers (rhPrimer GEN1 and rhPrimer GEN2). Both varieties contain a single RNA base to allow cleavage by RNase H. The difference between these two types of primers is that the cleaved region (blocking domain) on the Generation 1 primer is made of DNA bases only, whereas in the Generation 2 primer, there are C3 spacers for the purpose of stabilizing the blocking domain in the presence of a high-fidelity DNA polymerase with 3’ exonuclease function. Upon using the Generation 1 primers, Crissman et al. found an approximately 10-fold decrease in the unwanted small PCR product. Generation 2 primers gave even better PCR results, without any detectable small unwanted product.
When titering the antibody products from CHO cells, the researchers observed that desired products obtained after using rhPCR Generation 2 primers were increased compared to desired products obtained after using standard primers. Crissman et al. observed that their total antibody titers had an average increase of 56 μg/mL over their results from standard primers. In addition, from the total, more antigen-binding positive antibodies were obtained after use of rhPCR Generation 2 primers than standard primers, showing that the rhPCR primers had increased the amplification reaction’s sensitivity.
Rather than performing Sanger sequencing of the PCR products, Crissman et al. used well-specific index primers from IDT for next generation sequencing to tag the VH and VL amplicons in each of the wells. NGS showed that use of rhPCR primers in the previous experiments had increased the number of full-length antibody sequences. This was helpful not only because (as shown in the previous experiments) more of the desired antibodies had been synthesized, but also because this improved the NGS itself. The increased number of full-length sequences allowed the sequencing run to be more efficient, so that more samples could be processed in the same amount of time.
Crissman et al. concluded that rhPCR primers gave much better results than standard primers because they reduced formation of primer dimers. Therefore, falsely positive antibody titers were eliminated, and instead, an increase in the synthesis of desired antigen-binding positive antibodies took place. This approach also increased the full-length VH and VL sequencing reads. Even though the rhPCR primers were more expensive than standard primers, these many improvements in the experimental results increased the efficiency so that the overall cost of desired antibody production went down. Crissman et al. also concluded that their NGS approach to sequencing saved half the time that would have been required for Sanger sequencing and also cut the total sequencing costs to about 1/3 of what they would have been with Sanger sequencing.