PALINDROMIC SEQUENCE-TARGETED (PST) PCR, VERSION 2: AN ADVANCED METHOD FOR HIGH-THROUGHPUT TARGETED GENE CHARACTERIZATION AND TRANSPOSON DISPLAY
| dc.contributor.author | Kalendar, Ruslan | |
| dc.contributor.author | Shustov, Alexandr V. | |
| dc.contributor.author | Schulman, Alan H. | |
| dc.date.accessioned | 2021-08-12T08:31:20Z | |
| dc.date.available | 2021-08-12T08:31:20Z | |
| dc.date.issued | 2021-06-22 | |
| dc.description.abstract | 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. | en_US |
| dc.identifier.citation | Kalendar, R., Shustov, A. V., & Schulman, A. H. (2021). Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display. Frontiers in Plant Science, 12. https://doi.org/10.3389/fpls.2021.691940 | en_US |
| dc.identifier.issn | 1664-462X | |
| dc.identifier.uri | https://www.frontiersin.org/articles/10.3389/fpls.2021.691940/full | |
| dc.identifier.uri | https://doi.org/10.3389/fpls.2021.691940 | |
| dc.identifier.uri | http://nur.nu.edu.kz/handle/123456789/5681 | |
| dc.language.iso | en | en_US |
| dc.publisher | Frontiers Media S.A. | en_US |
| dc.relation.ispartofseries | Frontiers in Plant Science;22 June 2021 | https://doi.org/10.3389/fpls.2021.691940 | |
| dc.rights | Attribution-NonCommercial-ShareAlike 3.0 United States | * |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-sa/3.0/us/ | * |
| dc.subject | amplified fragment length polymorphism (AFLP) | en_US |
| dc.subject | biodiversity | en_US |
| dc.subject | genome walking | en_US |
| dc.subject | palindrome | en_US |
| dc.subject | restriction site | en_US |
| dc.subject | transposable elements (TE) | en_US |
| dc.subject | transposon display (TD) | en_US |
| dc.subject | Type of access: Open Access | en_US |
| dc.title | PALINDROMIC SEQUENCE-TARGETED (PST) PCR, VERSION 2: AN ADVANCED METHOD FOR HIGH-THROUGHPUT TARGETED GENE CHARACTERIZATION AND TRANSPOSON DISPLAY | en_US |
| dc.type | Article | en_US |
| workflow.import.source | science |