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RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells
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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643965/ https://www.ncbi.nlm.nih.gov/pubmed/33152031 http://dx.doi.org/10.1371/journal.pone.0241803 |
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author | Crissman, John Lin, Yuhao Separa, Kevin Duquette, Madeleine Cohen, Michael Velasquez, Candyd Cujec, Thomas |
author_facet | Crissman, John Lin, Yuhao Separa, Kevin Duquette, Madeleine Cohen, Michael Velasquez, Candyd Cujec, Thomas |
author_sort | Crissman, John |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7643965 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-76439652020-11-16 RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells Crissman, John Lin, Yuhao Separa, Kevin Duquette, Madeleine Cohen, Michael Velasquez, Candyd Cujec, Thomas PLoS One Research Article 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. Public Library of Science 2020-11-05 /pmc/articles/PMC7643965/ /pubmed/33152031 http://dx.doi.org/10.1371/journal.pone.0241803 Text en © 2020 Crissman et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Crissman, John Lin, Yuhao Separa, Kevin Duquette, Madeleine Cohen, Michael Velasquez, Candyd Cujec, Thomas RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells |
title | RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells |
title_full | RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells |
title_fullStr | RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells |
title_full_unstemmed | RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells |
title_short | RNase H-dependent PCR enables highly specific amplification of antibody variable domains from single B-cells |
title_sort | rnase h-dependent pcr enables highly specific amplification of antibody variable domains from single b-cells |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7643965/ https://www.ncbi.nlm.nih.gov/pubmed/33152031 http://dx.doi.org/10.1371/journal.pone.0241803 |
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