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  • Kalendar, Ruslan; Shustov, Alexandr; Schulman, Alan (2021)
    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.
  • Kalendar, Ruslan; Shustov, Alexandr; Seppänen, Mervi Mirjam; Schulman, Alan Howard; Stoddard, Frederick Lothrop (2019)
    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.