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

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dc.contributor University of Helsinki, Crop Science Research Group en
dc.contributor University of Helsinki, Helsinki Institute of Sustainability Science (HELSUS) en
dc.contributor University of Helsinki, Institute of Biotechnology en
dc.contributor University of Helsinki, Helsinki Institute of Sustainability Science (HELSUS) en
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 en
dc.identifier.issn 2045-2322
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 en
dc.subject 1183 Plant biology, microbiology, virology en
dc.subject ASYMMETRIC INTERLACED PCR en
dc.subject FAST PRIMER en
dc.subject AMPLIFICATION en
dc.subject FRAGMENTS en
dc.subject FASTPCR en
dc.subject TOOLS en
dc.subject PROBE en
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.description.version Peer reviewed
dc.identifier.doi https://doi.org/10.1038/s41598-019-54168-0
dc.type.uri info:eu-repo/semantics/other
dc.type.uri info:eu-repo/semantics/publishedVersion
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