Browsing by Subject "FAST PRIMER"

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  • Ghonaim, Marwa; Kalendar, Ruslan; Barakat, Hoda; Elsherif, Nahla; Ashry, Naglaa; Schulman, Alan (2020)
    Maize is one of the world’s most important crops and a model for grass genome research. Long terminal repeat (LTR) retrotransposons comprise most of the maize genome; their ability to produce new copies makes them efficient high-throughput genetic markers. Inter-Retrotransposon-Amplified Polymorphisms (IRAPs) were used to study the genetic diversity of maize germplasm. Five LTR retrotransposons (Huck, Tekay, Opie, Ji, and Grande) were chosen, based on their large number of copies in the maize genome, whereas polymerase chain reaction primers were designed based on consensus LTR sequences. The LTR primers showed high quality and reproducible DNA fingerprints, with a total of 677 bands including 392 polymorphic bands showing 58% polymorphism between maize hybrid lines. These markers were used to identify genetic similarities among all lines of maize. Analysis of genetic similarity was carried out based on polymorphic amplicon profiles and genetic similarity phylogeny analysis. This diversity was expected to display ecogeographical patterns of variation and local adaptation. The clustering method showed that the varieties were grouped into three clusters differing in ecogeographical origin. Each of these clusters comprised divergent hybrids with convergent characters. The clusters reflected the differences among maize hybrids and were in accordance with their pedigree. The IRAP technique is an efficient high-throughput genetic marker-generating method.
  • Kalendar, Ruslan; Raskina, Olga; Belyayev, Alexander; Schulman, Alan (2020)
    Retrotransposable elements are widely distributed and diverse in eukaryotes. Their copy number increases through reverse-transcription-mediated propagation, while they can be lost through recombinational processes, generating genomic rearrangements. We previously identified extensive, structurally uniform retrotransposon groups in which no member contains the gag, pol, or env internal domains. Because of the lack of protein-coding capacity, these groups are non-autonomous in replication, even if transcriptionally active. The Cassandra element belongs to the non-autonomous group called terminal-repeat retrotransposons in miniature (TRIM). It carries 5S RNA sequences with conserved RNA polymerase (pol) III promoters and terminators in its long terminal repeats (LTRs). Here, we identified multiple extended tandem arrays of Cassandra retrotransposons within different plant species, including ferns. At least 12 copies of repeated LTRs (as the tandem unit) and internal domain (as a spacer), giving a pattern that resembles the cellular 5S rRNA genes, were identified. Cytogenetic analysis revealed the specific chromosomal pattern of the Cassandra retrotransposon with prominent clustering at and around 5S rDNA loci. The secondary structure of the Cassandra retroelement RNA is predicted to form super-loops, in which the two LTRs are complementary to each other and can initiate local recombination, leading to the tandem arrays of Cassandra elements. The array structures are conserved for Cassandra retroelements of different species. We speculate that recombination events similar to those of 5S rRNA genes may explain the wide variation in Cassandra copy number. Likewise, the organization of 5S rRNA gene sequences is very variable in flowering plants; part of what is taken for 5S gene copy variation may be variation in Cassandra number. The role of the Cassandra 5S sequences remains to be established.
  • 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.
  • Vuorinen, Anssi L.; Kalendar, Ruslan; Fahima, Tzion; Korpelainen, Helena; Nevo, Eviatar; Schulman, Alan H. (2018)
    Wild emmer wheat (Triticum turgidum ssp. dicoccoides) is the wild ancestor of all cultivated tetraploid and hexaploid wheats and harbors a large amount of genetic diversity. This diversity is expected to display eco-geographical patterns of variation, conflating gene flow, and local adaptation. As self-replicating entities comprising the bulk of genomic DNA in wheat, retrotransposons are expected to create predominantly neutral variation via their propagation. Here, we have examined the genetic diversity of 1 Turkish and 14 Israeli populations of wild emmer wheat, based on the retrotransposon marker methods IRAP and REMAP. The level of genetic diversity we detected was in agreement with previous studies that were performed with a variety of marker systems assaying genes and other genomic components. The genetic distances failed to correlate with the geographical distances, suggesting local selection on geographically widespread haplotypes (‘weak selection’). However, the proportion of polymorphic loci correlated with the population latitude, which may reflect the temperature and water availability cline. Genetic diversity correlated with longitude, the east being more montane. Principal component analyses on the marker data separated most of the populations.