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 synthes

SUBMITTER: Crissman J 

PROVIDER: S-EPMC7643965 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature