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Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display

Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of...

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Autores principales: Kalendar, Ruslan, Shustov, Alexandr V., Schulman, Alan H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258406/
https://www.ncbi.nlm.nih.gov/pubmed/34239528
http://dx.doi.org/10.3389/fpls.2021.691940
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author Kalendar, Ruslan
Shustov, Alexandr V.
Schulman, Alan H.
author_facet Kalendar, Ruslan
Shustov, Alexandr V.
Schulman, Alan H.
author_sort Kalendar, Ruslan
collection PubMed
description Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5′ tail attached to the sequence-specific primer and the other anneals to a different 5′ tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2–3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.
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spelling pubmed-82584062021-07-07 Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display Kalendar, Ruslan Shustov, Alexandr V. Schulman, Alan H. Front Plant Sci Plant Science Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are in proximity to a known sequence tag, is currently predominantly based on PCR. Recently developed PCR-based methods allow for combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets, thereby offering the rapidity and effectiveness of PCR. This study presents a methodological improvement to the previously described GW technique known as palindromic sequence-targeted PCR (PST-PCR). Like PST-PCR, this new method (called PST-PCR v.2) relies on targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. PST-PCR v.2 consists of two rounds of PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers; one anneals to a 5′ tail attached to the sequence-specific primer and the other anneals to a different 5′ tail attached to the PST primer. The key advantage of PST-PCR v.2 is the convenience of using a single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes approximately 2–3 h to produce the amplified PCR fragment, which contains a portion of a template flanked by the sequence-specific and capturing primers. PST-PCR v.2 is highly suitable for simultaneous work with multiple samples. For this reason, PST-PCR v.2 can be applied beyond the classical task of GW for studies in population genetics, in which PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Furthermore, the conditions for PST-PCR v.2 are easier to optimize, as only one sequence-specific primer is used. This reduces non-specific random amplified polymorphic DNA (RAPD)-like amplification and formation of non-templated amplification. Importantly, akin to the previous version, PST-PCR v.2 is not sensitive to template DNA sequence complexity or quality. This study illustrates the utility of PST-PCR v.2 for transposon display (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites. Frontiers Media S.A. 2021-06-22 /pmc/articles/PMC8258406/ /pubmed/34239528 http://dx.doi.org/10.3389/fpls.2021.691940 Text en Copyright © 2021 Kalendar, Shustov and Schulman. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Kalendar, Ruslan
Shustov, Alexandr V.
Schulman, Alan H.
Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display
title Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display
title_full Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display
title_fullStr Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display
title_full_unstemmed Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display
title_short Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display
title_sort palindromic sequence-targeted (pst) pcr, version 2: an advanced method for high-throughput targeted gene characterization and transposon display
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8258406/
https://www.ncbi.nlm.nih.gov/pubmed/34239528
http://dx.doi.org/10.3389/fpls.2021.691940
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