Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species

Nthangeni, Mulalo Bethuel; Ramagoma, Faranani; Tlou, Matsobane Godfrey; Litthauer, Derek

doi:10.1016/j.mimet.2004.11.021

Cited by 25 publications

(13 citation statements)

References 20 publications

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“…Thus, convenient PCR-based methods have been developed instead of construction and screening of genome library. Some classical methods include inverse PCR [2,[4][5][6], PCR of various adapters (vectorett or linker) [7][8][9][10][11], restrictionsite PCR [12][13][14], and TAIL-PCR [15][16][17][18][19][20], which have extensive applications to genome walking in various organisms including microorganisms.…”

Section: Introductionmentioning

confidence: 99%

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Luo

et al. 2010

Mol Biotechnol

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Acquisition of flanking sequence adjacent to a known DNA site is an important task in microbial genome-related research. In this study, we developed a new method containing two rounds of PCR followed by cloning and sequencing. Firstly, specific primer (SP) is added into the reaction system for primary locus-specific linear amplification, and then a complex long primer (CLP) is added into the cooled reaction system for only one cycle. Amplification products from the first round of PCR are directly purified without electrophoresis, diluted, and used as the templates of the second PCR. Secondly, one long specific primer (LSP) and one long base-fixed primer (LFP) are adopted. The amplicons are purified for cloning and sequencing. The achievement of specific amplification for long flanking region mainly depends on ingenious and precise settings of PCR programs, structure design of CLP primer, adding of CLP primer after specific linear amplification, concentration ratio of CLP and SP primer, applying long primers, etc. Through this method, we successfully performed the long PCR walkings (>1.5 Kb) on rpoB gene of Vibrio vulnificus, transposon-like gene of V. alginolyticus, and sto gene of V. cholerae. The method provides a robust and simple strategy for rapid amplification of long unknown DNA fragments from microbes.

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Section: Introductionmentioning

confidence: 99%

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Luo

et al. 2010

Mol Biotechnol

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“…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.…”

Section: Gw Methods and Resourcesmentioning

confidence: 99%

Genome walking in eukaryotes

et al. 2011

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Genome walking is a molecular procedure for the direct identification of nucleotide sequences from purified genomes. The only requirement is the availability of a known nucleotide sequence from which to start. Several genome walking methods have been developed in the last 20 years, with continuous improvements added to the first basic strategies, including the recent coupling with next generation sequencing technologies. This review focuses on the use of genome walking strategies in several aspects of the study of eukaryotic genomes. In a first part, the analysis of the numerous strategies available is reported. The technical aspects involved in genome walking are particularly intriguing, also because they represent the synthesis of the talent, the fantasy and the intelligence of several scientists. Applications in which genome walking can be employed are systematically examined in the second part of the review, showing the large potentiality of this technique, including not only the simple identification of nucleotide sequences but also the analysis of large collections of mutants obtained from the insertion of DNA of viral origin, transposons and transfer DNA (T-DNA) constructs. The enormous amount of data obtained indicates that genome walking, with its large range of applicability, multiplicity of strategies and recent developments, will continue to have much to offer for the rapid identification of unknown sequences in several fields of genomic research.

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“…The subsequent singlestrand amplification with a single specific primer to enrich the targeted template strand was followed by a second conventional PCR with nested specific primer and an anchoring cassette primer (Nthangeni et al, 2005).…”

Section: Methods Applying Modification Of Dna Terminimentioning

confidence: 99%

Novel genes retrieved from environmental DNA by polymerase chain reaction: Current genome-walking techniques for future metagenome applications

Kotik

2009

Journal of Biotechnology

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Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species

Cited by 25 publications

References 20 publications

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

A Novel and Simple PCR Walking Method for Rapid Acquisition of Long DNA Sequence Flanking a Known Site in Microbial Genome

Genome walking in eukaryotes

Novel genes retrieved from environmental DNA by polymerase chain reaction: Current genome-walking techniques for future metagenome applications

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