methods Archives - Page 2 of 2

Method: New and improved multiplex PCR

Categories: methods, Summary for non-specialists

Tags: methods, PCR, Plant Methods, summary for non-specialists

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Published on: August 30, 2012

Highlighted article: Daxing Wen and Chuqing Zhang (2012) Universal Multiplex PCR: a novel method of simultaneous amplification of multiple DNA fragments. Plant Methods 8:32 (Online preview) doi: 10.1186/1746-4811-8-32

Background

Multiplex PCR allows amplification of multiple targets in a single PCR experiment. It is possible to amplify several sections of a single template, or to amplify different templates using a number of primer sets. If there are multiple primers in a reaction, it can be difficult optimise the PCR reaction to maximise the efficiency of every primer, and it is likely that some cross-hybridisation and mis-priming will occur.

Figure 3B from Wen and Zhange (2012). A comparison of multiplex PCR (Lanes 1-4) and universal multiplex PCR (lanes 5-8), using the same primers with universal adaptors. The band intensity from traditional PCR is very variable, but it is consistently strong when the universal adaptors are used.

Image credit: BioMed Central

The Method

Wen and Zhang from Shandong Agricultural University have devised a way around the inconveniences of multiplex PCR to develop a universal multiplex PCR method. ‘Universal adaptors’ are linked to specific primers, making the annealing temperature of the adaptor-primer structures 70°C. (more…)

Resource: Dataset of Arabidopsis genes with a loss-of-function mutant phenotype

by Charis Cook

Categories: methods, resource, Summary for non-specialists

Tags: gene, methods, phenotype, resources

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Published on: August 21, 2012

Highlighted article: J. Lloyd and D. Meinke (2012) A Comprehensive Dataset of Genes with a Loss-of-Function Mutant Phenotype in Arabidopsis thaliana. Plant Physiology, January 2012 pp.111.192393.

In plant science, many published papers involve at least one loss-of-function mutant. A huge number of mutant Arabidopsis lines exist in labs all around the world, detailed in as many journal articles. Now however the genotype and phenotype information for loss-of-function Arabidopsis mutants is stored one place: a dataset assembled by Johnny Lloyd and David Meinke of Oklahoma State University.

Lloyd and Meinke painstakingly went through TAIR, their own database SeedGenes.org, and PubMed to find 2400 Arabidopsis thaliana genes with a loss-of-function mutant phenotype. Out of necessity, the database excludes the effects of under- or over- expression of genes.

The phenotypic effects of gene knock-outs were classified into four groups: essential, morphological, cellular-biochemical, and conditional. The groups were divided into classes reflecting the developmental stage or organ where the phenotype manifests itself, and further divided into subsets which specify the characteristic affected by the phenotype, for example ‘pigmentation’, ‘gamerophyte defective’, and ‘stomata, trichomes’.

The dataset is found in the supplementary data of the paper. Supplemental Table 2 is the complete dataset. On tab 1 the dataset is sorted by locus number and includes 19 columns of information on the gene and the mutant phenotype. This information encompasses the classification of the phenotype, a description of the phenotype, and a reference to the lab in which the research was carried out. (more…)

Transcription factor-like effectors (TALEs)

by Charis Cook

Categories: methods, plant pathogens, Summary for non-specialists, synthetic biology

Tags: methods, plant pathogens, synthetic biology, TALEs

Comments: 2 Comments

Published on: August 8, 2012

Ubud, Bali by Mee Lin Woon; DNA Sequence by schulergd. Via stock.chng

Background

Xanthomonas spp. are plant pathogens that modulate their host’s gene expression in order to facilitate infection. They do this using transcription activator-like effectors (TALEs). Two domains are conserved in TALEs: an N-terminus, required for type III secretion into the plant cells; and a C-terminus with transcription factor activity. In the middle is a set of tandem repeats of amino acids, which mediates binding to host DNA.

As the binding and effector domains of TALEs can be customised, the possibility of using them for molecular and synthetic biology has been explored for some time. They have been used to change gene expression in plants, yeast, and even human cells.

TALEs have been adapted by researchers to make TALE nucleases (TALENs) – TALEs attached to a FOK1 nuclease domain. TALENs work in pairs that flank either side of the target site so that the nuclease domains meet at the point of cleavage. The nucleases cause a double-stranded DNA break, which is fixed imperfectly, causing an insertion or deletions.

In May this year, a paper was published demonstrating the huge impact TALEs could have on agriculture. Li et al. prove that transcription activator-like effector nucleases (TALENs) can be used to render rice resistant to the major agricultural pathogen, Xanthomonas oryzae pv. Oryzae (Xoo). (more…)

Identifying mutations in Arabidopsis – a faster, cheaper method

by Charis Cook

Categories: methods, Summary for non-specialists

Tags: forward genetics, genetics, methods, mutations, phenotype, Plant Methods

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Published on: July 30, 2012

Highlighted paper: Liu, McCormack and Sheen (2012) Targeted parallel sequencing of large genonmic regions for identifying mutations in Arabidopsis. Plant Methods 8:12

Kun-hsiang Liu, Matthew McCormack and Jen Sheen from Harvard have developed a PCR-based method of identifying mutations in Arabidopsis. It is cheaper and easier than traditional methods of identifying mutations, using bench-top PCR and a new user-friendly method of bioinformatics analysis using web-based resource Galaxy. Liu et al. estimate that using their method to identify a mutation mapped to a 550kb genomic region will cost roughly US$500, a fraction of the usual ten thousand dollar cost of currently used methods of mutant identification.

Liu et al. tested the new method of identifying mutations by searching for new nitrogen response genes. They generated an Arabidopsis thaliana line in which LUCIFERASE was driven by the promoter for nitrogen response marker NIR. Using EMS-mutagenesis, the team made 25 000 mutant NIR:LUC lines and identified seedlings that were nitrate insensitive (nis) or showed nitrate constitutive response (ncr).

When the lines were made, the phenotypes were identified and a second generation was grown. Three second generation lines – ncr1, nis1 and nis2 – were selected for further investigation.

Liu et al. used their novel TPSeq method to locate the mutations causing the ncr1, nis1 and nis2 phenotypes. (more…)

New TAIR10-compatible CDF files and review of RNA labelling methods

by Charis Cook

Categories: Summary for non-specialists

Tags: agronomics, methods, microarray, summary for non-specialists, TAIR

Comments: No Comments

Published on: July 17, 2012

A current paper in Plant Methods assessed the pros and cons of two RNA labeling methods for AGRONOMICS1 tiling arrays, concluding that random priming is more suitable for organelle transcriptome analysis as it can label non-polyadenylated transcripts effectively. They also generated new TAIR-10 based CDF files, which can be used to re-analyse existing AGRANOMICS1 CEL files. The new CDFs can be accessed here.

First of all, the authors gave an overview of the AGRONOMICS1 tiling array. It contains all the probes from the traditionally used Affymetrix ATH1 array, but has additional probes which mean the AGRONOMICS1 array yields expression data for over 7000 more genes, around a third of the genome. 90% of annotated genes on the TAIR9 database are on the array. Mitochondrial and chloroplast genomes are completely represented, and sRNA, tRNA and miRNA can also be detected. The AGRONOMICS1 array has probes that represent both strands of the entire Arabidopsis genome, allowing epigenetic profiling. The quality is comparable to that of the ATH1 array.

Müller et al. compared the GeneChip© IVT express kit, an oligo-dT based RNA labeling technique, with the GeneChip© whole transcript (WT) Sense Target Labeling Assay which uses random hexamers tagged with T7 promotor sequences. Both kits are from Affymetrix, Santa Carla, CA. (more…)

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Weeding the Gems

Method: New and improved multiplex PCR

Resource: Dataset of Arabidopsis genes with a loss-of-function mutant phenotype

Online resources for sharing and viewing data

Transcription factor-like effectors (TALEs)

Identifying mutations in Arabidopsis – a faster, cheaper method

New TAIR10-compatible CDF files and review of RNA labelling methods