Palindromic sequence-targeted (PST) PCR, version 2: an advanced method for high-throughput targeted gene characterization and transposon display

Show simple item record Kalendar, Ruslan Shustov, Alexandr Schulman, Alan 2021-06-19T12:25:01Z 2021-06-19T12:25:01Z 2021-06-22
dc.identifier.citation Kalendar , R , Shustov , A & Schulman , A 2021 , ' Palindromic sequence-targeted (PST) PCR, version 2: an advanced method for high-throughput targeted gene characterization and transposon display ' , Frontiers in plant science , vol. 12 , 691940 .
dc.identifier.other PURE: 163133659
dc.identifier.other PURE UUID: ab4104e6-21b4-4737-85f5-09cb5ee144bb
dc.identifier.other ORCID: /0000-0003-3986-2460/work/95992792
dc.identifier.other WOS: 000669845700001
dc.description.abstract Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that exist 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, which offer 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 hours 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, which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the corn (Zea mays) genome was used as a sequence tag during the transposon display procedure to characterize the Ac integration sites. en
dc.format.extent 12
dc.language.iso eng
dc.relation.ispartof Frontiers in plant science
dc.rights cc_by_nd
dc.rights.uri info:eu-repo/semantics/openAccess
dc.subject 11831 Plant biology
dc.subject genome walking
dc.subject transposon display (TD)
dc.subject palindrome
dc.subject restriction site
dc.subject amplified fragment length polymorphism (AFLP)
dc.subject transposable elements (TE)
dc.subject biodiversity
dc.subject JAVA WEB TOOLS
dc.subject IN-SILICO PCR
dc.subject FAST PRIMER
dc.subject FRAGMENTS
dc.subject ELEMENTS
dc.subject FASTPCR
dc.subject REMAP
dc.title Palindromic sequence-targeted (PST) PCR, version 2: an advanced method for high-throughput targeted gene characterization and transposon display en
dc.type Article
dc.contributor.organization Institute of Biotechnology
dc.contributor.organization Crop Science Research Group
dc.contributor.organization Department of Agricultural Sciences
dc.contributor.organization Viikki Plant Science Centre (ViPS)
dc.contributor.organization Biosciences
dc.description.reviewstatus Peer reviewed
dc.relation.issn 1664-462X
dc.rights.accesslevel openAccess
dc.type.version publishedVersion

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