s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis

The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor™) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.     

Soil warming accelerates decomposition of fine woody debris

Aims

There is evidence that increased N inputs to boreal forests, via atmospheric deposition or intentional fertilization, may impact negatively on ectomycorrhizal (ECM) fungi leading to a reduced flux of plant-derived carbon (C) back to the atmosphere via ECM. Our aim was to investigate the impact of N fertilization of a Pinus sylvestris (L.) forest stand on the return of recently photoassimilated C via the ECM component of soil respiration.

Methods

We used an in situ, large-scale, ¹³C-CO₂ isotopic pulse labelling approach and monitored the ¹³C label return using soil gas efflux chambers placed over three different types of soil collar to distinguish between heterotrophic (R_H), autotrophic (R_A; partitioned further into contributions from ECM hyphae and total R_A) and total (R_S) soil respiration.

Results

The impact of N fertilization was to significantly reduce R_A, particularly respiration via extramatrical ECM hyphae. ECM hyphal flux in control plots showed substantial spatial variability, resulting in mean flux estimates exceeding estimates of total R_A, while ECM contributions to R_A in N treated plots were estimated at around 30%.

Conclusion

Significant impacts on soil C cycling may be caused by reduced plant C allocation to ECM fungi in response to increased N inputs to boreal forests; ecosystem models so far lack this detail.     

Comparison of Texture Features Derived from Static and Respiratory-Gated PET Images in Non-Small Cell Lung Cancer

Background

PET-based texture features have been used to quantify tumor heterogeneity due to their predictive power in treatment outcome. We investigated the sensitivity of texture features to tumor motion by comparing static (3D) and respiratory-gated (4D) PET imaging.

Methods

Twenty-six patients (34 lesions) received 3D and 4D [¹⁸F]FDG-PET scans before the chemo-radiotherapy. The acquired 4D data were retrospectively binned into five breathing phases to create the 4D image sequence. Texture features, including Maximal correlation coefficient (MCC), Long run low gray (LRLG), Coarseness, Contrast, and Busyness, were computed within the physician-defined tumor volume. The relative difference (δ_3D-4D) in each texture between the 3D- and 4D-PET imaging was calculated. Coefficient of variation (CV) was used to determine the variability in the textures between all 4D-PET phases. Correlations between tumor volume, motion amplitude, and δ_3D-4D were also assessed.

Results

4D-PET increased LRLG ( = 1%–2%, p<0.02), Busyness ( = 7%–19%, p<0.01), and decreased MCC ( = 1%–2%, p<7.5×10⁻³), Coarseness ( = 5%–10%, p<0.05) and Contrast ( = 4%–6%, p>0.08) compared to 3D-PET. Nearly negligible variability was found between the 4D phase bins with CV<5% for MCC, LRLG, and Coarseness. For Contrast and Busyness, moderate variability was found with CV = 9% and 10%, respectively. No strong correlation was found between the tumor volume and δ_3D-4D for the texture features. Motion amplitude had moderate impact on δ for MCC and Busyness and no impact for LRLG, Coarseness, and Contrast.

Conclusions

Significant differences were found in MCC, LRLG, Coarseness, and Busyness between 3D and 4D PET imaging. The variability between phase bins for MCC, LRLG, and Coarseness was negligible, suggesting that similar quantification can be obtained from all phases. Texture features, blurred out by respiratory motion during 3D-PET acquisition, can be better resolved by 4D-PET imaging. 4D-PET textures may have better prognostic value as they are less susceptible to tumor motion.     

Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing

PCR is widely employed as the initial DNA amplification step for genetic testing. However, a key limitation of PCR-based methods is the inability to selectively amplify low levels of mutations in a wild-type background. As a result, downstream assays are limited in their ability to identify subtle genetic changes that can have a profound impact in clinical decision-making and outcome. Here we describe co-amplification at lower denaturation temperature PCR (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence. We replaced regular PCR with COLD-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold and identified new mutations in the genes encoding p53, KRAS and epidermal growth factor in heterogeneous cancer samples that had been missed by the currently used methods. For clinically relevant microdeletions, COLD-PCR enabled exclusive amplification and isolation of the mutants. COLD-PCR will transform the capabilities of PCR-based genetic testing, including applications in cancer, infectious diseases and prenatal identification of fetal alleles in maternal blood.     

MOSFET assessment of radiation dose delivered to mice using the Small Animal Radiation Research Platform (SARRP)

The Small Animal Radiation Research Platform (SARRP) is a novel isocentric irradiation system that enables state-of-the-art image-guided radiotherapy research to be performed with animal models. This paper reports the results obtained from investigations assessing the radiation dose delivered by the SARRP to different anatomical target volumes in mice. Surgically implanted metal oxide semiconductor field effect transistors (MOSFET) dosimeters were employed for the dose assessment. The results reveal differences between the calculated and measured dose of -3.5 to 0.5%, -5.2 to -0.7%, -3.9 to 0.5%, -5.9 to 2.5%, -5.5 to 0.5%, and -4.3 to 0% for the left kidney, liver, pancreas, prostate, left lung, and brain, respectively. Overall, the findings show less than 6% difference between the delivered and calculated dose, without tissue heterogeneity corrections. These results provide a useful assessment of the need for tissue heterogeneity corrections in SARRP dose calculations for clinically relevant tumor model sites.     

A fast analytic dose calculation method for arc treatments for kilovoltage small animal irradiators

Arc treatments require calculation of dose for collections of discrete gantry angles. The sampling of angles must balance between short computation time of small angle sets and the better calculation reliability of large sets. In this paper, an analytical formula is presented that allows calculation of dose delivered during continuous rotation of the gantry. The formula holds valid for continuous short arcs of up to about 30° and is derived by integrating a dose formula over gantry angles within a small angle approximation. Doses for longer arcs may be obtained in terms of doses for shorter arcs. The formula is derived with an empirical beam model in water and extended to inhomogeneous media. It is validated with experimental data obtained by applying arc treatment using kV small animal irradiator to a phantom of solid water and lung-equivalent material. The results are a promising step towards efficient 3D dose calculation and inverse planning purposes. In principle, this method also applies to VMAT dose calculation and optimization but requires extensions.