Haplotyping germline and cancer genomes with high-throughput linked-read sequencing.

Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.

Multiplexed target detection using DNA-binding dye chemistry in droplet digital PCR.

Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.

Process development and production of cGMP grade Melan-A for cancer vaccine clinical trials.

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body's immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%.

Conformationally preorganised hosts for anions using norbornane and fused [n]polynorbornane frameworks.

Norbornane and fused [n]polynorbornane frameworks are readily synthesised, can be tailored to a variety of predictable geometries and can be functionalised regiospecifically. As such, these highly preorganised scaffolds offer the supramolecular chemist an excellent starting point when designing hosts for specific guests. This feature article will highlight the evolution of our research from relatively simple norbornane based anion receptors to more sophisticated tetrathioureido functionalised fused [n]polynorbornane hosts.

Examples of regioselective anion recognition among a family of two-, three-, and four-"armed" bis-, tris-, and tetrakis(thioureido) [n]polynorbornane hosts.

A family of conformationally preorganized, [n]polynorbornane-based anion hosts 1a,b-6a,b have been synthesized. The series includes receptors with 4, 8, and 12 H-bond donors. Using (1)H NMR titration techniques, evaluation of the new hosts against a series of alkyl and aryl dicarboxylates as well as a range of phosphoanionic species has revealed that the tris(thioureido) hosts (in particular 3a) are capable of regioselectively binding dicarboxylates and pyrophosphate (H(2)PPi(2-)).

Methods for chromatographic removal of endotoxin.

Endotoxin removal is critical when producing therapeutic proteins in bacterial systems. This hydrophobic compound can be removed through chromatography or filtration, but presents unique challenges dependent upon protein composition as well as production scale. Here we present a robust method for endotoxin removal at the pilot production scale using fast protein liquid chromatography and buffers specifically engineered for endotoxin removal.

Expression and purification of cGMP grade NY-ESO-1 for clinical trials.

NY-ESO-1 is a cancer testis antigen expressed in numerous cancers. Initial tests have shown its efficacy as a cancer vaccine, stimulating the body's own immune response against the invading tumor. To produce enough material for phase I clinical trials, a process using current good manufacturing practices to produce clinical grade material was developed and executed. His-tagged NY-ESO-1 was expressed in C41DE3 Escherichia coli under control of the T-7 promoter. NY-ESO-1 was produced in a 20 L fed-batch fermentation utilizing a pH-stat control scheme. The protein was then purified from inclusion bodies using a three-column process that achieved a yield of over 3.4 g and endotoxin below the detection limit of 0.005 EU/µg protein.

Multiplex single nucleotide polymorphism genotyping utilizing ligase detection reaction coupled surface enhanced Raman spectroscopy.

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a clinically significant number of markers can be multiplexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are significant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity.

Recombinant immunotherapeutics: current state and perspectives regarding the feasibility and market.

Recombinant immunotherapeutics are important biologics for the treatment and prevention of various diseases. Immunotherapy can be divided into two categories, passive and active. For passive immunotherapy, the successes of antibody and cytokine therapeutics represent a promising future and opportunities for improvements. Efforts, such as cell engineering, antibody engineering, human-like glycosylation in yeast, and Fab fragment development, have led the way to improve antibody efficacy while decreasing its high manufacturing costs. Both new cytokines and currently used cytokines have demonstrated therapeutic effects for different indications. As for active immunotherapy, recently approved HPV vaccines have encouraged the development of preventative vaccines for other infectious diseases. Immunogenic antigens of pathogenic bacteria can now be identified by genomic means (reverse vaccinology). Due to the recent outbreaks of pandemic H1N1 influenza virus, recombinant influenza vaccines using virus-like particles and other antigens have also been engineered in several different recombinant systems. However, limitations are found in existing immunotherapeutics for cancer treatment, and recent development of therapeutic cancer vaccines such as MAGE-A3 and NY-ESO-1 may provide alternative therapeutic strategy.

Binding of the terephthalate dianion by di- tri- and tetrathiourea functionalised fused [3] and [5]polynorbornane based hosts.

The affinity of new di-, tri- and tetrathiourea functionalised fused [3] and [5]polynorbornane based hosts 1-6 towards terephthalate2- was proportional to the size of the preorganised cleft/cavity imparted by the polynorbornane scaffold. Receptors based on the [5]polynobornane framework had greater affinities for the anion due to a higher degree of host:guest size complementarity. Hosts 1-5 formed 1:1 host:guest complexes with the rigid dianion, yet remarkably, host 6 was found to bind two terephthalate guests.

Surface-enhanced Raman scattering based ligase detection reaction.

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye's Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform.

Size matters--strong binding of the terephthalate dianion by thiourea functionalised fused [n]polynorbornane hosts.

Remarkably strong binding of the new [5]polynorbornane based host to the terephthalate dianion is based on size complementarity of the preorganised binding cleft with the rigid dicarboxylate guest.

Steric and electronic factors influencing recognition by a simple, charge neutral norbornene based anion receptor.

Based on (1)H NMR studies, subtle electronic factors rather than pre-organisation dictate the binding stoichiometry of the new, norbornene based, anion hosts 1 and 2 with acetate, however, the binding of dihydrogenphosphate appears to be based solely on steric constraints.

Antibacterial activity of (-)-deoxypseudophrynaminol versus its racemate and derivatives.

(-)-Deoxypseudophrynaminol 1 possesses 43-fold greater antibacterial potency than the racemate toward Staphylococcus aureus, indicating that the (-)-enantiomer is the biologically active isomer in this assay. Comparison of the percent growth inhibition by derivatives of 1 indicates that prenylation of N8 and replacement of N1-methyl by methyl carbamate are detrimental to antibacterial potency. (-)-1 is a promising lead structure for the development of the novel hexahydropyrrolo[2,3-b]indole class of antibacterial agents.