Palindromic Sequence-Targeted (PST) PCR, Version 2: An Advanced Method for High-Throughput Targeted Gene Characterization and Transposon Display

Abstract

Genome walking (GW), a strategy for capturing previously unsequenced DNA fragments that are 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, thereby offering 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 h 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 (TD), which is a method to characterize inter- or intra-specific variability related to transposon integration sites. The Ac transposon sequence in the maize (Zea mays) genome was used as a sequence tag during the TD procedure to characterize the Ac integration sites.

Genetic Structure, Differentiation and Originality of Pinus sylvestris L. Populations in the East of the East European Plain

“…It would be ideal to use markers characterized by wide genome distribution and [17], above all, would be accessible for any species including those that have not been studied. Such DNA genetic markers include all PCR-based DNA fingerprinting method variants of the Random Amplified Polymorphic DNA (RAPD) method [18], such as Inter Simple Sequence Repeat (ISSR) [19], Palindromic Sequence-targeted (PST) PCR [20] and others [21]. This list can also be supplemented with methods based on interspersed repeat sequences in genomes, including a range of retrotransposon related elements [22].…”

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes

“…Thermal cycling conditions were selected based on recommendations described in Kalendar et al [ 16 , 17 ]. The thermal profile consists of two stages: a linear phase which uses a high annealing temperature to favor binding of the transgene-specific primer for initial template formation, and an exponential phase at a lower annealing temperature to favor binding of random primers to the transgene-containing template.…”

“…For decades genome-walking methods have been used to amplify unknown genomic sequences with varying degrees of success. Here, we utilized one of the more recent variations on this method from Kalendar et al [ 16 , 17 ] which was designed to alleviate some of the major bottlenecks that are commonly encountered in genome-walking PCR and also addresses some limitations of insertion-site finding methods. By combining this method with high-throughput sequencing, we show in an example using diploid Arabidopsis thaliana and allohexaploid Camelina sativa transgenic lines how detection of insertion sites in the T 1 generation of two stacked transgenic camelina lines can be quickly and easily characterized.…”

Editorial: Mobile Elements and Plant Genome Evolution, Comparative Analyzes and Computational Tools

“…Potential TEs also contribute to genome plasticity and have a dramatic impact on the genetic diversity and evolution of the wheat genome. Using transposon display (Kalendar et al, 2021) and genome-wide profiling analysis of insertional polymorphisms of transposable elements (Kalendar and Schulman, 2006), the authors discovered large genomic rearrangement events, such as deletions and introgressions in the wheat genome. Highthroughput bioinformatics with next-generation sequencing (NGS) were key tools in these studies (Vondrak et al, 2020).…”