RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells

Abstract

Immunization-based antibody discovery platforms require robust and effective protocols for the amplification, cloning, expression, and screening of antibodies from large numbers of B-cells in order to effectively capture the diversity of an experienced Ig-repertoire. Multiplex PCR using a series of forward and reverse primers designed to recover antibodies from a range of different germline sequences is challenging because primer design requires the recovery of full length antibody sequences, low starting template concentrations, and the need for all the primers to function under the same PCR conditions. Here we demonstrate several advantages to incorporating RNase H2-dependent PCR (rh-PCR) into a high-throughput, antibody-discovery platform. Firstly, rh-PCR eliminated primer dimer synthesis to below detectable levels, thereby eliminating clones with a false positive antibody titer. Secondly, by increasing the specificity of PCR, the rh-PCR primers increased the recovery of cognate antibody variable regions from single B-cells, as well as downstream recombinant antibody titers. Finally, we demonstrate that rh-PCR primers provide a more homogeneous sample pool and greater sequence quality in a Next Generation Sequencing-based approach to obtaining DNA sequence information from large numbers of cloned antibody cognate pairs. Furthermore, the higher specificity of the rh-PCR primers allowed for a better match between native antibody germline sequences and the VL/VH fragments amplified from single B-cells.

Fig 1. PCR amplification of VH fragments from single B-cells.

(A) Amplification of VH fragments using standard FR1 and J-region primers results in full-length product (arrowhead), as well as a truncated fragment of <150 bp in size (double arrowhead). (B) A representative gel of a series of VH fragments. (C) Recovery of cognate, full-length VL and VH antibody domains from single B-cells using framework 1 and J-region primers. The green box denoted recovery of cognate full length VL and VH fragments. Wells not receiving a B-cell are designated as water wells. (D) Ab titers following transient expression of cognate VL and VH pairs in CHO cells. Light and dark blue cells denote transfection and Octet controls, respectively. (E) Western blot analysis of antibodies containing either a full-length or a truncated VH fragment. Antibodies containing full-length or truncated VH fragments are indicated with a single or double arrowhead respectively.

Fig 2. IDT Blocked cleavable primers.

Design comparison of IDT’s Generation 1 versus Generation 2 Blocked Cleavable Primers for use in rh-PCR. After annealing to the target sequence, the thermostable RNase H2 enzyme cleaves at the single ribonucleotide releasing the Blocking Domain allowing for primer extension. The Generation 2 primer design represents an upgrade in the stability of the blocking group and was developed specifically for use with high fidelity DNA polymerases that contain a 3’ exonuclease function. (D–DNA base complimentary to target, M–DNA base that is a mismatch to target, r–RNA base complimentary to target, x–C3 Spacer.

Fig 3. Analysis of standard and rh-PCR Gen-1 and primers for recovery of antibody variable domains.

Amplification of VH regions using standard FR1 and J-regions primers or the rh-PCR Gen-1 variants as detected on an agarose gel (A) and by capillary electrophoresis (B). Full length VH fragment (arrowhead), small PCR product (double arrowhead) and unused Primer Pool (PP) are indicated. (C) quantification of truncated PCR products as a percentage of total DNA in each well following amplification of VH fragments using rh-PCR Gen-1 primers (D) Sensitivity of the rh-PCR Gen-1 primers to the 3’ exonuclease activity of the HiFi Phusion DNA polymerase and Exonuclease I.

Fig 4. Characterization of rh-PCR Gen-2 primers.

(A) Incubation of the VL rh-PCR Gen-2 primer pool with Exonuclease I or VII. (B) Amplification of VH fragments using standard FR1 and J-region PCR primers and the rh-PCR Gen-1 and rh-PCR Gen-2 derivatives. The electropherogram is on the left and a gel pseudo-image on the right. UM- CE Upper Marker, LM- CE Lower Marker, VH- variable region PCR product, PP- unused Primer Pool, U- Unwanted products.

Fig 5. Characterization of VL and VH amplicons following amplification with rh-PCR Gen-2 primers.

(A) Agarose gels of standard primers (top row) or rh-PCR Gen-2 primers (bottom row). Gels of the VL fragments are on the left and of the VH fragments on the right. (B) Electropherograms comparing a series of amplicons produced using standard primers (left) to those produced using rh-PCR Gen-2 primers (right). Truncated PCR products (U- Unwanted products) and unused primers (PP- Primer Pool) are denoted by arrowheads. (C) Comparison of wells lacking a full length VL (left) or VH (right) products amplified with either standard or rh-PCR Gen-2 primers. Truncated PCR products and unused primers are captured by a parenthesis and a single arrowhead, respectively. UM- CE Upper Marker, LM- CE Lower Marker, VH- variable region PCR product, PP- unused Primer Pool, U- Unwanted products.

Fig 6. IgG titers following amplification of VL and VH regions using standard FR1 and J-region primers or their rh-PCR Gen-2 derivatives.

Full-length VL and VH amplicon pairs using the standard primers are denoted by the green box (A). Antibody titers following VL and VH PCR using the standard primers (B), or the rh-PCR Gen-2 primers (C).

Fig 7. Ability of antibodies cloned from amplicons using standard primers or the rh-PCR Gen-2 variants to bind antigen in an ELISA format.

Graphed by well in descending order of signal. Blue bars correspond to PCR #2 with standard primers, while orange bars correspond to PCR #2 with rh-PCR Gen-2 primers for the same samples. Blue and red dotted lines denote the average absorbance for unsuccessful PCR wells using the standard and rh-PCR Gen-2 primers, respectively. Newly identified antigen+ wells from rh-PCR Gen-2 supernatants are indicated by vertical arrows.

Fig 8. Next Generation Sequencing of amplicons derived from standard and rh-PCR Gen-2 primers.

(A) schematic representation of the workflow. (B) Agarose gels of VH regions amplified (1^st PCR) from miniprep DNA cloned from either the standard nested primers (left) or the rh-PCR Gen 2 primers (right). Full length VH fragment (arrowhead), small PCR product (double arrowhead). (C) Fraction of reads for the major clonotype in each well matching the expected germline sequences in the first 21 nucleotides of framework 1. The distribution of sequences is compared using the standard nested PCR primers (left) or the rh-PCR primers (right) for both VL (turquoise) and VH (red) sequences.