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Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parall...

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Detalles Bibliográficos
Autores principales: Murgha, Yusuf E., Rouillard, Jean-Marie, Gulari, Erdogan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986247/
https://www.ncbi.nlm.nih.gov/pubmed/24733454
http://dx.doi.org/10.1371/journal.pone.0094752
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author Murgha, Yusuf E.
Rouillard, Jean-Marie
Gulari, Erdogan
author_facet Murgha, Yusuf E.
Rouillard, Jean-Marie
Gulari, Erdogan
author_sort Murgha, Yusuf E.
collection PubMed
description Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.
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spelling pubmed-39862472014-04-15 Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries Murgha, Yusuf E. Rouillard, Jean-Marie Gulari, Erdogan PLoS One Research Article Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification. Public Library of Science 2014-04-14 /pmc/articles/PMC3986247/ /pubmed/24733454 http://dx.doi.org/10.1371/journal.pone.0094752 Text en © 2014 Murgha 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Murgha, Yusuf E.
Rouillard, Jean-Marie
Gulari, Erdogan
Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
title Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
title_full Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
title_fullStr Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
title_full_unstemmed Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
title_short Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
title_sort methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3986247/
https://www.ncbi.nlm.nih.gov/pubmed/24733454
http://dx.doi.org/10.1371/journal.pone.0094752
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