TY - JOUR
T1 - Palindromic sequence-targeted (PST) PCR
T2 - a rapid and efficient method for high-throughput gene characterization and genome walking
AU - Kalendar, Ruslan
AU - Shustov, Alexandr V.
AU - Seppänen, Mervi M.
AU - Schulman, Alan H.
AU - Stoddard, Frederick L.
N1 - Funding Information:
This study was funded under Academy of Finland Decision 314961. The study was also financed in part by a grant from the Ministry of Education and Science of Kazakhstan (АР05130607, AP05130266).
Publisher Copyright:
© 2019, The Author(s).
Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.
PY - 2019/12/1
Y1 - 2019/12/1
N2 - 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.
AB - 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.
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U2 - 10.1038/s41598-019-54168-0
DO - 10.1038/s41598-019-54168-0
M3 - Article
C2 - 31776407
AN - SCOPUS:85075605697
SN - 2045-2322
VL - 9
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 17707
ER -