Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking

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dc.contributor.author Kalendar, Ruslan
dc.contributor.author Shustov, Alexandr
dc.contributor.author Seppänen, Mervi Mirjam
dc.contributor.author Schulman, Alan Howard
dc.contributor.author Stoddard, Frederick Lothrop
dc.date.accessioned 2019-11-29T12:14:01Z
dc.date.available 2019-11-29T12:14:01Z
dc.date.issued 2019-11-27
dc.identifier.citation Kalendar , R , Shustov , A , Seppänen , M M , Schulman , A H & Stoddard , F L 2019 , ' Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking ' , Scientific Reports , vol. 9 , no. 17707 , 17707 . https://doi.org/10.1038/s41598-019-54168-0
dc.identifier.other PURE: 123694121
dc.identifier.other PURE UUID: 8ae468df-c45f-4633-bc76-18918e62824d
dc.identifier.other ORCID: /0000-0002-8097-5750/work/65308332
dc.identifier.other ORCID: /0000-0003-3986-2460/work/65308608
dc.identifier.other WOS: 000499210200001
dc.identifier.uri http://hdl.handle.net/10138/307667
dc.description.abstract Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3’ ends. Upstream of the palindromes there is a degenerate sequence (8-12 nucleotides long); defined adapters are present at the 5’-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods. en
dc.format.extent 11
dc.language.iso eng
dc.relation.ispartof Scientific Reports
dc.rights cc_by_nd
dc.rights.uri info:eu-repo/semantics/openAccess
dc.subject 1183 Plant biology, microbiology, virology
dc.subject ASYMMETRIC INTERLACED PCR
dc.subject FAST PRIMER
dc.subject AMPLIFICATION
dc.subject FRAGMENTS
dc.subject FASTPCR
dc.subject TOOLS
dc.subject PROBE
dc.title Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking en
dc.type Article
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 Helsinki Institute of Sustainability Science (HELSUS)
dc.contributor.organization Forages for the future
dc.contributor.organization Plant Production Sciences
dc.contributor.organization Institute of Biotechnology
dc.contributor.organization Legume science
dc.description.reviewstatus Peer reviewed
dc.relation.doi https://doi.org/10.1038/s41598-019-54168-0
dc.relation.issn 2045-2322
dc.rights.accesslevel openAccess
dc.type.version publishedVersion

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