PrimerIdent---一个基于web的保守引物设计工具

Bioinformation

Volume 5

Issue 2

open access Hypothesis

PrimerIdent: A web based tool for conserved primer design

Alberto M Pessoa1,2*, Susana Pereira 1,2, Jorge Teixeira1,2

1

University of Porto, Faculty of Sciences, Biology Department, Edifício FC4, Rua do Campo Alegre, s/n, 4169-007 Porto, Portugal; 2BioFIG - Center for Biodiversity, Functional & Integrative Genomics, Plant Molecular Biology & Biotechnology Lab, University of Porto, Portugal; Alberto M Pessoa - Email addresses: pessoa.am@fc.up.pt *Corresponding author

Received May 28, 2010; accepted June 09, 2010; published July 06, 2010

Abstract:

result, which offers a list of all possible primers that meet the user criteria, with a colour-code identity to each sequence in the alignment. The software here presented is a simple and intuitive web based tool that is suitable for distinguishing very similar nucleotide sequences, such as isozymes-coding sequences, to enable the conserved primer design across multiple species, necessary for approaches that rely on knowing if a primer is suitable for a certain set of pre-aligned sequences, to design a specific primer to a certain sequence variation, or a combination thereof. This extremely useful software can, therefore, be used as a tool for the specific amplification of individual members of multigenic families across related species and also to evaluate the differential expression of isogenes for a given species.

Availability: http://primerident.up.pt

Keywords: Primer design, conserved primers, multiple-purposed primers, gene-specific primers

Background:

Polymerase chain reaction (PCR) is a robust, flexible and fast technique that is essential to the modern Molecular Biology and is currently used in many applications. One of the fundamental assets for a successful PCR reaction is the quality and reliability of the oligonucleotide primer design.Designing primers in conserved regions of related nucleotide sequences is of great importance for molecular biologists as it allows the usage of the same primer to successfully obtain a PCR product across multiple and/or understudied organisms. Softwares that can design conserved primers using multiple alignments have been developed, and are free to use, such as Primaclade [1].

PrimerIdent, however, uses the concept of conserved primer design for a different purpose: PCR (or RT-PCR) amplification of a specific isozyme-coding sequence.Isozymes are multiple forms of enzymes from the same class arising from genetically determined differences in primary structure and frequently sharing a great identity amongst them. As a consequence, it is very difficult to design primers that effectively differentiate between related genes that share a high degree of similarity. The difficulty lies on choosing primers that are specific for one sequence coding a particular isozyme and simultaneously less identical to similar sequences coding other members of the same gene family.In order to design a suitable primer for a nucleotide sequence coding a specific isozyme, it is necessary to ensure that the primer is designed for a exclusive region for that isozyme. This primer design approach makes the conserved primer design tools described above unsuitable.One way of doing this, albeit time-consuming and complex, is to find these primers using a trial/error method, with the aid of BLAST (Basic Local Alignment Search Tool – [2]), by comparing the match score of the different published sequences, and therefore minimizing the risk of amplifying more than one nucleotide coding sequence for a type of isozyme at the same time.

ISSN 0973-2063 (online) 0973-8894 (print) Bioinformation 5(2): 52-54 (2010)

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Such approach proves to be even more complex if the studied organism doesn’t have any nucleotide sequences published for a particular multigene family. This means that, in this case, it is necessary to find a primer that is i) conserved across multiple organisms phylogenetically close to the organism studied, and simultaneously ii) specific for a given isozyme. This isozyme specificity should also be consistent across those related organisms.To circumvent these complex tasks, PrimerIdent was developed aiming to automate this primer design and selection process, in a given nucleotide sequence alignment providing an intuitive, easy to interpret graphical output.

Input:

PrimerIdent was implemented as a Perl-CGI script that uses Primer3 core [3] as the primer design engine and accepts a FASTA alignment of no more than 8 sequences with a maximum length of 10 kbp at a time. Figure 1 shows the web frontend. The website is located at http://primerident.up.pt, hosted by the University of Porto, Portugal. In an interactive user interface, the menu on the left (A) provides basic information about PrimerIdent, which includes an introduction section and application examples, as well as a F.A.Q. (Frequently Asked Questions) section and contact information.In order to use PrimerIdent, the user pastes the FASTA sequence alignment into the textbox (B). This alignment can be made in programs such as ClustalW [4] or MEGA4 [5]. Before submitting the alignment for analysis, the user has to select one of the sequences to be considered the template for primer design, normally corresponding to a cDNA of the target isozyme of the most closely related species (C), and choose between left and right primer design (D). As the Primer3 core is used as the primer design engine, the user can also modify the different criteria that the Primer3 engine should use to pick all possible primers for that template sequence (E). It is also possible to sort the found primers by sequence location or output order (i.e. the same order by which the Primer3 core finds them - F).

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