![]() ![]() ![]() Various PCR-based GW approaches have been used to capture DNA fragments from representatives of all kingdoms of life 2. A review and the systematics of PCR-based GW methods have been presented 2. In turn, applying the degenerate oligonucleotides in PCR with complex templates (genomic DNA) is often accompanied by nonspecific amplification this is a driving force behind the development of new methods, such as PST-PCR as described here. a protein alignment for a similar target, requires the use of degenerate oligonucleotides as walking primers. Often, minimal a priori knowledge about the target, e.g. The best walking primer for a particular application reflects a good guess, or a working hypothesis about the unknown target DNA. the ability to generate a GW product) and specificity (avoiding generation of unwanted products). In principle, all significant improvements to PCR-based GW technologies have been aimed at controlling the balance between sensitivity (i.e. A paired companion to the SSP is a walking primer that is intended to solve the problem of annealing (with the greatest achievable specificity) to an unknown target. PCR-based GW can be employed when it is possible to select at least one sequence-specific primer (SSP) that anneals to the DNA of interest. The first applications of PCR as an alternative to hybridization were reported in the late 1980s 1. PCR-based methods are currently predominant among GW techniques because they are rapid and their use avoids construction of large libraries. use of a sequenced fragment as a probe for hybridization). The initial GW technology also utilized libraries and screening (e.g. ![]() Before the advent of next-generation sequencing (NGS), capturing of unknown DNA was based on GW or screening of genomic libraries. Genome walking (GW) refers to a collection of methods that capture unknown (unsequenced) genomic regions that are contiguous with and adjacent to a known DNA sequence. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods. 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 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. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Upstream of the palindromes there is a degenerate sequence (8–12 nucleotides long) defined adapters are present at the 5′-termini. The PST primers have palindromic sequences at their 3′-ends. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. 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. ![]()
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