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June 2012

MyTaq™ - How to Take Your PCR From 11 Hours to TWO

By Bioline: The PCR Company 14 June 2012 No comments

Fides LayWe caught Fides Lay in the lab on the day of a basketball game between UCLA and crosstown arch-rival USC. As a former UCLA undergrad now working on her PhD at USC, she never wants to miss a game between the two. Unfortunately, the long hours in the lab don’t always allow her to be home in time to watch. At least, that’s how it used to be for her. Thanks to Bioline’s MyTaq HS Mix, now she doesn’t miss a game.

In Peter Jones’ lab at USC, Fides is part of a team studying the epigenetic regulation of cancer. And as anyone who ever worked on epigenetics knows, you need to be a master of PCR to get any results at all. When you need to amplify bisulfite converted DNA to measure methylation status, you work with small samples that often have been digested with several enzymes, with DNA that is fragmented and damaged.

For the longest time the lab amplified bisulfite converted DNA with a tedious protocol; tedious because of the long set-up with using Taq from a previous supplier, DMSO, and other components added one at a time, and extremely slow cycling with a PCR protocol that was 7-11 hours long.

Anything could go wrong at any time, and the result was impossible to predict. Simply using a different thermocycler with a different ramping speed could cause the reaction to fail. And once you found out, it would be too late to fix because every new attempt takes a whole other day. Since the lab usually clones and sequences the PCR fragments, there was always still the risk that no clones would have inserts. Hard to predict, and that meant starting over.

All that changed when Fides first tried Bioline’s MyTaq HS Mix, an easy, all-in-one mix that contains the enzyme, dNTPs, buffer and all optimizers. There’s no need to add any DMSO, it works right away on almost all templates. And it's fast! Reactions are done in less than two hours, even on bisulfite converted DNA, with highly consistent results, and always with nice bands. The PCR products are much easier to clone, and on the rare occasion that something does go wrong, there’s still time to redo the experiment AND get the samples off to pyrosequencing the same day.

Now Fides has time to run multiple experiments and redo anything that goes wrong, all in time to get home, kick up her feet and watch the game.

Bioline Scholar Monthly: May 2012 Round-up – Plant Epigenetics

By Bioline: The PCR Company 7 June 2012 No comments

Focus on Plant Epigenetics

2012 marks the 150th anniversary of the publication of Charles Darwin's first botanical book, on the fertilization of orchids (1862), wherein he described pollen grains and outlined his evolutionary principles with respect to plant research. Gregor Mendel was also coming to the end of this famous pea study (1856-1863). The 'green biotech-revolution' of the 21st century can be traced back to these classical breeding experiments; however, with the use of molecular biology - and in particular the information we have gained from the sequencing of many plant species - there has been an unprecedented development in every field of plant science in the last few years.

Plants are crucial to human society as they provide everything from habitat to other essentials for survival such as food, oxygen, and medicines, while also creating and preserving the essential soils required for this cycle to continue. Plants are also required for other, non-essential items such as cosmetics and an almost infinite range of products for people and society. As human population increases, so too does the our dependence on plants and, consequently, the need to understand plants and their potential in the coming decades is increasing, leading to greater emphasis on the calls for basic plant biological research.

Bioline offers a number of reagents that have helped further the study and understanding of plants. This month's edition of Bioline Scholar focuses on the use of Bioline reagents and kits in the field of plant epigenetic research.

SensiFAST™ SYBR & Fluorescein Kit

The results in this exciting paper from the Gregor Mendel Institute of Molecular Plant Biology in Austria suggest that DMS11 (defective in meristem silencing 11) provides the missing ATPase function for DMS3 and that these proteins cooperate in the RNA-directed DNA methylation pathway to promote transcriptional repression. GHKL ATPases are thus emerging as new players in epigenetic regulation in plants and mammals.

Lorkovic C., et al. Current Biol. 22: 1–6 (2012) - Involvement of a GHKL ATPase in RNA-Directed DNA Methylation in Arabidopsis thaliana [PDF]

SensiMix™ SYBR No-ROX Kit

Jones and colleagues studied the flowering of Eucalyptus globulus subsp. globulus, an important forestry species in temperate parts of the world. The globulus homologues, FLOWERING LOCUS T (FT) and LEAFY (LFY) were examined shown to form part of the flower initiation pathway in Eucalyptus but do not regulate the observed differences in anthesis time.

Jones R. C., et al. Australian J. Botany 59 (8): 756-769 (2012) - Expression of a FLOWERING LOCUS T homologue is temporally associated with annual flower bud initiation in Eucalyptus globulus subsp. globulus (Myrtaceae)

hmdCTP (5-hydroxymethyl-2'-deoxycytidine 5'-triphosphate)

Yao's study asks how well the Arabidopsis thaliana Variant in Methylation 1 (VIM1) protein, an essential factor in maintaining 5-cytosine methylation (5mC) homeostasis and epigenetic silencing in this plant, recognizes 5-hydroxymethyl-cytosine (5hmC). They found that 5hmC may contribute to VIM-mediated passive loss of cytosine methylation in vivo during Arabidopsis DNA replication.

Yao Q., et al. Protein Expression and Purification 83 (1): 104–111 (2012) - Heterologous expression and purification of Arabidopsis thaliana VIM1 protein: In vitro evidence for its inability to recognize hydroxymethylcytosine, a rare base in Arabidopsis DNA

MangoTaq™ DNA Polymerase

The characteristic expression pattern of AtBMI1C is the result of a complex epigenetic process. The results show the orchestrated interplay of different epigenetic mechanisms in regulating gene expression throughout development, shedding light on the current hypotheses for the origin and mechanism of imprinting in plant endosperm.

Bratzel, F., et al. Mol. Plant 5 (1): 260-269 (2012) - Regulation of the new Arabidopsis imprinted gene AtBMI1C requires the interplay of different epigenetic mechanisms

BioMix™

A targeted epigenetic response to a specific environmental stress in a physiologically important pathway is reported, suggesting epigenetic regulation of stomatal development that allows for anatomical and phenotypic plasticity, and may help to explain at least some of the resilience to fluctuating relative humidity.

Tricker P. J., et al. J. Expt. Botany doi: 10.1093/jxb/ers076 (2012) - Low relative humidity triggers RNA-directed de novo DNA methylation and suppression of genes controlling stomatal development.

BIO-X-ACT™ Short DNA Polymerase

Next-generation sequencing of barcoded amplicon mixtures was used to reliably sample all alleles of homeologous loci in polyploid species and successfully investigate phylogenetic relationships among species, as well as to investigate phylogeographic hypotheses. This next-generation sequencing method is more affordable than and at least as reliable as bacterial cloning. It could be applied to any experiment involving sequencing of amplicon mixtures.

Griffin P. C., et al. BMC Biol. 9: 19 (2011) - A next-generation sequencing method for overcoming the multiple gene copy problem in polyploid phylogenetics, applied to Poa grasses

In Arabidopsis thaliana, the ability to flower is mainly related to a floral repressor, FLOWERING LOCUS C (FLC), which is regulated through the vernalization pathway. This paper shows that the regulation of CiMFL gene expression in time and space and in relation to environmental conditions is only partially conserved with respect to FLC in A. thaliana. A model for flowering repression by CiMFL is proposed.

Locascio A., et al. New Phytologist 182 (3): 630–643 (2009) - Characterization of a MADS FLOWERING LOCUS C-LIKE (MFL) sequence in Cichorium intybus: a comparative study of CiMFL and AtFLC reveals homologies and divergences in gene function

BIOTAQ™ DNA Polymerase

Raphanus sativus is a widely cultivated member of the family Brassicaceae, for which genomic resources are available only to a limited extent in comparison to many other members of the family. The genetic map developed here is expected to provide a standard map for the genetics, genomics, and molecular breeding of R. sativus as well as of related species.

Shirasawa K., et al. DNA Res. 18 (4): 221-232. (2011) - An EST-SSR Linkage Map of Raphanus sativus and Comparative Genomics of the Brassicaceae

This paper presents the first reported survey of bacteria in floral nectar from a natural plant community. The results showed that bacteria are common inhabitants of floral nectar plants and their communities are characterized by low species richness and moderate phylogenetic diversity, with most isolates belonging to the Gammaproteobacteria, showing osmotolerance, catalase activity and the ability to grow under microaerobiosis, to overcome factors limiting their survival in nectar.

Álvarez-Pérez S., et al. FEMS Microbiol. Ecol. 80 (3): 591–602 (2012) - Zooming-in on floral nectar: a first exploration of nectar-associated bacteria in wild plant communities.

BioMix™

The most economically important species is O. ficus indica, cultivated both for fruits and cladodes, but little is known about their ancestries and levels of genetic diversity. This study increases our knowledge of the variability among some of the most diffused Opuntia cultivated accessions and also points to the inconsistencies of previous taxonomical genotype assignments that were based solely on morphological characteristics.

Caruso M., et al. Plant Syst. Evol. 290 (1-4): 85-97 (2010) - Microsatellite markers help to assess genetic diversity among Opuntia ficus indica cultivated genotypes and their relation with related species

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