![]() The adaptor is composed of two synthetic oligonucleotides with uneven lengths, so that the adaptor has the protruding 5′-end. The molecular design of the proprietary adaptor is important, as the design provides for increased specificity. To increase chances of success, the manufacturer recommends simultaneously performing the GW on four libraries, each produced with a different restriction enzyme. Restriction endonucleases yield blunt ends to which synthetic adaptors are subsequently ligated. The GenomeWalker GW process starts with the creation of “libraries” of DNA fragments, of which each library consists of digestion products of starting DNA. The GenomeWalker Kits are ligation-based, requiring ligation of adaptors to blunt-ended fragments of target DNA the approach is called “suppression PCR” because the generation of side products is suppressed during PCR. With regard to these systems, a comparison of the modern methods for capturing unknown DNA illustrates a diversity of operable approaches. Two widely published commercial technologies are the GenomeWalker Kits (Clontech & Takara) and the APAgene GW kit (Bio S&T) 2. More recently, GW-like techniques have proven valuable for studies in which DNA capture itself is not the ultimate goal such studies include population genetics and diversity studies on populations and species 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17.Ĭurrent PCR-based GW methods are more elaborate than the protocols initially used, and some patented techniques are available for researchers in the form of commercial GW kits. 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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