دانلود رایگان مقاله انگلیسی پیمایش ژنوم در یوکاریوت ها به همراه ترجمه فارسی
| عنوان فارسی مقاله: | پیمایش ژنوم در یوکاریوت ها |
| عنوان انگلیسی مقاله: | Genome walking in eukaryotes |
| رشته های مرتبط: | زیست شناسی، ژنتیک، بیوشیمی، علوم سلولی و مولکولی و میکروبیولوژی |
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| نشریه | وایلی – Wiley |
| کد محصول | f313 |
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بخشی از مقاله انگلیسی: Introduction Identification of unknown nucleotide sequences flanking already characterized DNA regions can be pursued by a number of different PCR-based methods commonly known as genome walking (GW). In times of high-throughput DNA sequencing technologies, when more than 1000 genomes have been completely sequenced, the development of GW strategies can appear as an out-of-date laboratory activity. Nevertheless, papers describing applications of GW methods and improvements of several available strategies continue to be published with a steady positive trend. Reasons for such constant interest can be found both in the relatively low difficulty of the different strategies, which do not require expensive equipment or highly trained personnel, and in the increasing possibilities of applying GW methods to eukaryotic genomes. Furthermore, in some highly sophisticated applications, GW strategies have recently been combined with pyrosequencing technology allowing the production of hundreds of thousands of sequences per single experiment and opening new application areas for GW. Review articles on GW are not numerous. After a first paper by Hengen dated 1995 [1], where a limited number of strategies were compared, a complete survey of the available strategies was published by Hui et al. [2] more than a decade ago. Recently, two reviews have been dedicated to the description of GW strategies and their applications, but limited to microorganisms [3,4]. This review is intended to provide the missing information by describing the application of GW techniques to eukaryotic genomes. Numerous reports can be found in which such technology has been successfully used, avoiding in many cases the time-consuming process of the construction and screening of large genomic libraries. A first section gives a general overview of the available GW methods, classified according to the basic strategy adopted. Most of these methods can be easily executed in any molecular biology laboratory. In addition, a list of commercial resources (kits and customer services) is also provided. A second part of the paper deals with the different applications of GW. Main areas of interest have been identified and the results obtained for specific applications are reported. Owing to the large diffusion of GW methods, this survey cannot by any means be complete. We have done our best to show the huge potential of these methods and we apologize to colleagues whose work has not been reported. GW methods and resources GW methods GW methods differ in the strategies adopted to obtain the substrate for a final PCR step, in which a primer specific for the known sequence (sequence specific primer) is coupled with a primer dictated by the specific strategy of walking (walking primer). In Table 1, the basic GW methods and related improvements that have been developed are listed. Methods are classified into three different groups according to their first (and sometimes conditioning) step: restriction-based (R-GW) methods, primer-based (P-GW) methods and extension-based (E-GW) methods. GW methods using a combination of two of the basic strategies are catalogued according to the first step of the procedure. Most of the methods listed in Table 1 have already been critically reviewed [1–4] and are not described further in this paper. Only more recent methods, together with those previously not reviewed, are examined in this report. These methods are highlighted in Table 1. Graphical representations of the strategies at the basis of GW methods are schematically reported in Fig. 1. R-GW methods require a preliminary digestion of the genomic DNA by suitable restriction enzymes, whose sites must be located at a proper distance from the boundary between known and unknown sequences (not too far in order to allow subsequent PCR amplifi- cation; not too close to avoid amplification of short fragments). Restriction fragments can then be either self-circularized or ligated to specifically designed adaptors. In the first case, the sub-group of the inverted-PCR (I-PCR) methods, first described by Triglia et al. [5], is obtained (Table 1). An improvement to this strategy, named rolling circle I-PCR, has recently been reported [8]. In this case circularized genomic DNA restriction molecules are subjected to rolling circle amplification by using random hexamers and employing the stranddisplacement property of Phi29 polymerase. In the latter case, a wide range of methods have been developed starting from the first strategy named single-specific-primer PCR [9]. These methods are catalogued according to Tonooka and Fujishima [3] as ‘cassette PCR’, for the use of double-stranded DNA linkers to ligate to genomic DNA restriction fragments. We prefer the term ‘cassette PCR’ instead of ‘ligation mediated PCR’, which is sometimes also used to indicate these methods ([7,21,22,28,44], for example), since the term ‘ligation mediated’ has been used since 1989 to indicate one of the first GW strategies, here classified among the E-GW methods (Table 1). Once the cassette has been ligated to genomic DNA restriction fragments, generating what is commonly known as the GW library, a PCR amplification of the region encompassing the boundary between the known and unknown sequences can be carried out using a sequence specific primer and a cassette specific primer. One major concern in the cassette PCR based methods is the background of non-specific products due to the cassette specific primer. To overcome this problem a number of tricks have been devised, such as those adopted in vectorette PCR [11], capture PCR [13] and other strategies reported below. The strategy known as transfer DNA (T-DNA) fingerprinting PCR [20] adopted for GW an amplified fragment length polymorphism method developed for studying the number of Agrobacterium tumefaciens T-DNA insertions in transgenic plants. Amplified fragments corresponding to T-DNA ⁄ plant DNA junctions, identifiable thanks to a labelled T-DNA specific primer, are eluted from polyacrylamide gel, re-amplified and sequenced. |