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

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http://hdl.handle.net/10138/307667

Lähdeviite

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

Julkaisun nimi: Palindromic sequence-targeted (PST) PCR: a rapid and efficient method for high-throughput gene characterization and genome walking
Tekijä: Kalendar, Ruslan; Shustov, Alexandr; Seppänen, Mervi Mirjam; Schulman, Alan Howard; Stoddard, Frederick Lothrop
Muu tekijä: University of Helsinki, Crop Science Research Group
University of Helsinki, Helsinki Institute of Sustainability Science (HELSUS)
University of Helsinki, Institute of Biotechnology
University of Helsinki, Helsinki Institute of Sustainability Science (HELSUS)
Päiväys: 2019-11-27
Kieli: eng
Sivumäärä: 11
Kuuluu julkaisusarjaan: Scientific Reports
ISSN: 2045-2322
URI: http://hdl.handle.net/10138/307667
Tiivistelmä: 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.
Avainsanat: 1183 Plant biology, microbiology, virology
ASYMMETRIC INTERLACED PCR
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AMPLIFICATION
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