DMSO and Betaine Greatly Improve Amplification of GC-Rich Constructs in De Novo Synthesis

In Synthetic Biology, de novo synthesis of GC-rich constructs poses a major challenge because of secondary structure formation and mispriming. While there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation. Therefore, to overcome these limitations in researching GC-rich genes and their non-coding elements, we explored the use of DMSO and betaine in two conventional methods of assembly and amplification. For this study, we compared the polymerase (PCA) and ligase-based (LCR) methods for construction of two GC-rich gene fragments implicated in tumorigenesis, IGF2R and BRAF. Though we found no benefit in employing either DMSO or betaine during the assembly steps, both additives greatly improved target product specificity and yield during PCR amplification. Of the methods tested, LCR assembly proved far superior to PCA, generating a much more stable template to amplify from. We further report that DMSO and betaine are highly compatible with all other reaction components of gene synthesis and do not require any additional protocol modifications. Furthermore, we believe either additive will allow for the production of a wide variety of GC-rich gene constructs without the need for expensive and time-consuming sample extraction and purification prior to downstream application

Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy

Genome-Wide Analyses of Recombination Prone Regions Predict Role of DNA Structural Motif in Recombination

HapMap findings reveal surprisingly asymmetric distribution of recombinogenic regions. Short recombinogenic regions (hotspots) are interspersed between large relatively non-recombinogenic regions. This raises the interesting possibility of DNA sequence and/or other cis- elements as determinants of recombination. We hypothesized the involvement of non-canonical sequences that can result in local non-B DNA structures and tested this using the G-quadruplex DNA as a model. G-quadruplex or G4 DNA is a unique form of four-stranded non-B DNA structure that engages certain G-rich sequences, presence of such motifs has been noted within telomeres. In support of this hypothesis, genome-wide computational analyses presented here reveal enrichment of potential G4 (PG4) DNA forming sequences within 25618 human hotspots relative to 9290 coldspots (p<0.0001). Furthermore, co-occurrence of PG4 DNA within several short sequence elements that are associated with recombinogenic regions was found to be significantly more than randomly expected. Interestingly, analyses of more than 50 DNA binding factors revealed that co-occurrence of PG4 DNA with target DNA binding sites of transcription factors c-Rel, NF-kappa B (p50 and p65) and Evi-1 was significantly enriched in recombination-prone regions. These observations support involvement of G4 DNA in recombination, predicting a functional model that is consistent with duplex-strand separation induced by formation of G4 motifs in supercoiled DNA and/or when assisted by other cellular factors

Social Relationships and Mortality Risk: A Meta-analytic Review

In a meta-analysis, Julianne Holt-Lunstad and colleagues find that individuals' social relationships have as much influence on mortality risk as other well-established risk factors for mortality, such as smoking

Measurement of the WZ production cross section in pp collisions at root s=7 and 8 TeV and search for anomalous triple gauge couplings at root s=8 TeV

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