Restriction enzyme site-directed amplification PCR: A tool to identify regions flanking a marker DNA

González-Ballester, David; Montaigu, Amaury de; Galván, Aurora; Fernández, Emilio

doi:10.1016/j.ab.2005.01.031

Cited by 102 publications

(112 citation statements)

References 18 publications

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“…To identify the region flanking the marker inserted, both the RESDA-PCR [25] and inverse PCR after digestion with an enzyme having a unique restriction site in the marker gene, [26] were successfully used. Most of the mutants were solved by inverse PCR and those mutants recalcitrant to inverse PCR were successfully solved by RESDA.…”

Section: Discussionmentioning

confidence: 99%

“…RESDA-PCR basically involves using specific primers for the marker gene together with degenerated primers, designed on the basis of the presence of frequent restriction sites randomly distributed in the microalgal genome [25]. A schematic representation of the RESDA strategy with indication of the annealing sites for the primers is shown in Figure 1.2.…”

Section: Identification Of the Genomic Regions Flanking The Insertionmentioning

confidence: 99%

“…The only exception was mutant D45 that seems to have several copies of the marker gene. Two different methods were used to determine the genomic DNA flanking the inserted marker gene: RESDA-PCR [25] or inverse PCR over ligation [26].…”

Section: Identification Of the Genomic Regions Flanking The Insertionmentioning

confidence: 99%

“…Restriction enzyme site-directed amplification PCR [25], RESDA-PCR, is modified from Thermal asymmetric interlaced TAIL-PCR [31] and basically involves using specific primers for the marker gene together with degenerated primers. The design of these degenerated primers is based on the presence of frequent restriction sites randomly distributed in the microalgal genome.…”

Section: Resda Pcrmentioning

confidence: 99%

“…Mutants which have higher violaxanthin content (B20), higher pigments concentration (A7), lower chlorophyll content (C13) and total lack of chlorophylls and carotenoids (C2), together with three mutants that synthesize small quantities of zeaxanthin in response to high light stress (Z7, Z34 and Z198), have been isolated and physiologically characterized. The insertion site in 15 of the selected mutants has been characterized by RESDA PCR (Polymerase Chain Reaction) [25] or inverse-nested PCR [26], contributing to increase the information generated by previous photosynthetic functional genomic analysis in Chlamydomonas [20].…”

Section: Introductionmentioning

confidence: 99%

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Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

Vilà¹,

Díaz-Santos²,

Vega³

et al. 2013

Genomics Discov

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In the present work, a large collection of mutants was obtained by insertional mutagenesis from 704 (cw15 Arg7+ Nia1 : Ars mt+ ) strain of Chlamydomonas reinhardtii, using a linearized fragment of the pSI03 plasmid. About 2100 insertional mutants resistant to the antibiotic paromomycin were isolated and screened to identify Chlamydomonas reinhardtii mutants: i) sensitive to high light, ii) with an altered pigment composition and iii) with an irregular response to high light stress. The insertion site in 15 of the selected mutants was amplified by Restriction enzyme site-directed amplification (RESDA) PCR or inverse PCR. Cloning, sequencing and alignment of the amplified DNA with the Chlamydomonas database allowed the identification of the region adjacent to the insertion in most of the mutants studied. We observed that in 77 % of the transformants analysed, insertion took place in intragenic regions. Almost half of the mutants (46 %) had insertions in genome loci with unknown functions. Among disrupted genes with known functions, we found genes involved in a great diversity of functions, from flagella motion to regulatory or signal transduction processes, suggesting that the sensitivity to high light and the synthesis of pigments are complex and very regulated processes.

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Section: Discussionmentioning

confidence: 99%

Section: Identification Of the Genomic Regions Flanking The Insertionmentioning

confidence: 99%

Section: Identification Of the Genomic Regions Flanking The Insertionmentioning

confidence: 99%

Section: Resda Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

Vilà¹,

Díaz-Santos²,

Vega³

et al. 2013

Genomics Discov

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Subtractive hybridization magnetic bead capture: A new technique for the recovery of full‐length ORFs from the metagenome

Meyer

Burton

Cowan

2007

Biotechnology Journal

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A number of PCR-based (i.e., sequence dependent) techniques are available for the recovery of full-length functional genes from genomic DNA preparations. These include semi-nested PCR, TAIL PCR and inverse PCR [5,6]. The implementation of these techniques may be technically challenging, particularly for metagenomic samples [7]. To expedite and simplify gene recovery, we have adapted magnetic bead capture and subtractive hybridization techniques [8] for the specific recovery of fulllength ORFs. The combination of these techniques paves the way for more effective recovery of full-length functional genes from metagenomic DNA samples.In this study we sought to develop a more direct and rapid gene recovery technique to avoid some of the problems associated with gene-specific PCR of metagenomic samples. The use of gene-specific labeled magnetic beads in a subtractive hybridization assay has been adapted from Jacobsen [8], who demonstrated the microscale detection of Pseudomonas fluorescens genomic DNA in soil using a lux gene fragment as the immobilized probe.We modified this technique to allow the specific recovery of a target gene rather than the entire genome. Broadly, multiple target genes are PCR-amplified from metagenomic DNA using gene-specific degenerate primers. The partial gene products of this amplification process (the 'driver DNA'; Box 1) are then immobilized to streptavidin-covered magnetic beads and used as hybridization probes to recover 'full-length' genes from metagenomic DNA samples (Fig. 1).The first step in the DNA preparation is the introduction of adapter priming sites. Adapter sequences were reconstructed and ligated to the 3´-A-tailed metagenomic DNA fragments ('tester DNA': Box 1). To ensure efficient adapter ligation, a directional cloning strategy was employed. The adapters were designed to be partially single stranded at the 5´ end, forcing the incorporation of the adapter priming sites in the correct orientation. Prior to hybridization, the priming sites must be reconstructed by blunt-ending the adaptor 5´ overhang.There is potentially no limitation to the nature of the environmental samples that may be used. However, consideration must be given to the chemical and biochemical properties of the sample, and the way in which these It is now widely accepted that classical microbiological methods provide only limited access to the true microbial biodiversity (less than 1%) [1]. The desire to access a higher proportion of the metagenome [2] has led to the development of efficient environmental nucleic acid extraction technologies and to a range of sequence-dependent and sequence-independent gene discovery techniques [3]. These methods [2, 4] avoid many of the limitations of culture-dependent gene targeting.

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Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin‐2 support an “on‐demand” model for Chlamydomonas ciliary length control

Patel,

Griffin,

Olson

et al. 2024

Cytoskeleton

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Most cells tightly control the length of their cilia. The regulation likely involves intraflagellar transport (IFT), a bidirectional motility of multi‐subunit particles organized into trains that deliver building blocks into the organelle. In Chlamydomonas, the anterograde IFT motor kinesin‐2 consists of the motor subunits FLA8 and FLA10 and the nonmotor subunit KAP. KAP dissociates from IFT at the ciliary tip and diffuses back to the cell body. This observation led to the diffusion‐as‐a‐ruler model of ciliary length control, which postulates that KAP is progressively sequestered into elongating cilia because its return to the cell body will require increasingly more time, limiting motor availability at the ciliary base, train assembly, building block supply, and ciliary growth. Here, we show that Chlamydomonas FLA8 also returns to the cell body by diffusion. However, more than 95% of KAP and FLA8 are present in the cell body and, at a given time, just ~1% of the motor participates in IFT. After repeated photobleaching of both cilia, IFT of fluorescent kinesin subunits continued indicating that kinesin‐2 cycles from the large cell‐body pool through the cilia and back. Furthermore, growing and full‐length cilia contained similar amounts of kinesin‐2 subunits and the size of the motor pool at the base changed only slightly with ciliary length. These observations are incompatible with the diffusion‐as‐a‐ruler model, but rather support an “on‐demand model,” in which the cargo load of the trains is regulated to assemble cilia of the desired length.

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Restriction enzyme site-directed amplification PCR: A tool to identify regions flanking a marker DNA

Cited by 102 publications

References 18 publications

Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

Isolation and characterization of pigment deficient insertional mutants in the chlorophyte Chlamydomonas reinhardtii

Subtractive hybridization magnetic bead capture: A new technique for the recovery of full‐length ORFs from the metagenome

Distribution and bulk flow analyses of the intraflagellar transport (IFT) motor kinesin‐2 support an “on‐demand” model for Chlamydomonas ciliary length control

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