Amplification Of Azospirillum Sp. Jg3 GLPD Gene Fragment Using Degenerate Primers Generated By Web-Based Tools
2025; RELX Group (Netherlands); Linguagem: Inglês
10.2139/ssrn.5030987
ISSN1556-5068
AutoresStalis Norma Ethica, Mohammad Kasfu Hammi, Puji Lestari, Endang Semiarti, Jaka Widada, Tri Joko Raharjo,
Tópico(s)Identification and Quantification in Food
ResumoFaculty of Mathematics and Natural Science, Gadjah Mada University, Kaliurang Street, Sekip Utara, Yogyakarta 55281, Indonesia. *Corresponding author: trijr_mipa@ugm.ac.id ABSTRACT Keywords: Degenerate primer, primaclade, in silico, colony PCR, Azospirillum sp. JG3 INTRODUCTION To answer many questions of evolutionary developmental biology, it is necessary to identify genes which could be responsible for the studied traits (Žlůvova, 2007). PCR amplification of homologous genes using degenerate primers followed by direct sequencing is a standard approach to generate a data set for evolutionary analysis (Regier and Shi, 2005). Degenerate primers are easy and cheap to produce regular unique primers, are useful for amplifying several related genomic sequences, and have been used in various applications (Linhart and Shamir 2005). However, degenerate primer design can be still very difficult because of codon degeneracy and the additional degeneracy needed to represent multiple codons at a position in the alignment (Žlůvova, 2007). Not only are the calculating tasks heavy, a ranking mechanism for optimization is also very sophisticated. Therefore, the computational aid on primer design is a critical issue in bioinformatics (Chen, et al., 2003). Numerous computer programs are now available allowing users to design oligo-nucleotides nearly without preliminary knowledge. Most of these programs are available for free and straightforward to use from convenient web-interfaces (Christensen, et al., 2008). Though not all of these resources are well maintained, some of them, including Primaclade (http://www.umsl.edu/services/kellogg/primaclade.html), are ofhigh quality. Primaclade is a web-based application that accepts a multiple species nucleotide alignment file as input and identifies a set of polymerase chain reaction (PCR) primers that will bind across the alignment. Primaclade iteratively runs the Primer3 application for each alignment sequence and collates the results (Gadberry, et al., 2005). Along with CODEHOP (Rose, et al., 2003) Primaclade is one of classical methods for degenerate primer design based on multiple global alignments to identify clear blocks of conserved regions (Gorron, et al, 2010). In Silico PCR analysis program (http://In Silico.ehu.es/) is a useful and efficient complementary method to ensure primer specificity for an extensive range of PCR applications. This in-silico PCR method can assist in the selection of newly designed primers, identify potential mismatches in the primer binding sites and avoid the amplification of unwanted amplicons so that potential problems can be prevented before any “wet bench” experiment (Yu and Zhang, 2011). Recently the application of Primaclade was successful in the evolutionary development study of genus Begonia (Neale, et al., 2006) and the genomic island identification study of E.coli and Shigella flexneri (Ou, et al., 2006). In Silico application was also prospering in the multi-copy gene family expression study of Plasmodium falciparum (Bachmann, et al., 2009) and pseudogene analysis study of human and mouse (Sun, et al., 2012). However, a strategy combining of Primaclade and In Silico techniques in glpD gene identification study of genus Azospirillum has not been implemented. Azospirillum sp. JG3 strain is a member of Azospirillum genus, which members are frequently associated with root and rhizosphere of a large number of agriculturally important crops and cereals (Saharan and Nehra 2011). However, lack of media for selective isolation or techniques for specific detection or identification limit the exploration of these rhizobacteria. This has motivated many researchers to design a genus-specific oligo-nucleotide primer pair which could assist in rapid detection of species of the genus Azospirillum by means of PCR-specific amplification (Lin, et al., 2011). Despite of its poor genetic and genomic information, the Gram-negative Azospirillum sp. JG3 strain was known to have ability to produce industry enzymes, lipase and amylase (Lestari et al., 2009; Zusfahair and Ningsih, 2012). This research is an attempt to reveal the genetic properties of Azospirillum sp. JG3 bacteria by identifying its glpD gene encoding glycerol-3-phosphate dehydrogenase essential for the aerobic growth of bacteria in glycerol or glycerol-3-phosphate (Choi, et al., 1989). The objective of our study was to propose degenerate primers designed using Primaclade which could be used to design primers when only a limited number of sequences with high global similarity to their suspected homologues available. We also demonstrated the use of In Silico web-based tools to simulate a virtual PCR using the obtained primers that can avoid potential problems in our in-vitro experiment. We aimed to obtain reliable oligo-nucleotides that can amplify glpD gene fragment of strain Azospirillum sp. JG3. We show that using a limited number of aligned sequences the amplification of DNA sequences with similarity to glycerol-metabolism related genes in other member of Azospirillum genus (possibly homologues) is highly possible using Primaclade. Experimentally, the combination use of Primaclade and In Silico Primaclade and In Silico web-based tools were used as a strategy to obtain the correct-size PCR amplicon targeting a fragment of gene encoding glycerol-3-phosphate dehydrogenase (glpD) of Azospirillum sp. JG3. The bacterial strains are soil, Gram-negative PGPR (Plant-Growth Promoting Rhizobacteria) isolated from an agricultural land in Purwokerto, Central Java, Indonesia, which have ability to produce several commercial enzymes. The aim is to obtain a pair of reliable degenerate primers from a limited number of glpD sequences from other Azospirilla retrieved in GenBank using bioinformatics approach. We demonstrated degenerate primer design that led to successful PCR amplification corresponding to the targeted DNA fragment. Homology analysis showed that the obtained DNA fragment is 61% and 99% similar to sn-glycerol-3-phosphate dehydrogenase genes of Azospirillum brasilense and Stenotrophomonas maltophili respectively. ARTICLE INFO Received 11. 9. 2013 Revised 29. 10. 2013 Accepted 30. 10. 2013 Published 1. 12. 2013 Short communication
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