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www. deltadot.com deltaDOT Ltd. Bessemer Building Prince Consort Road South Kensington London SW7 2BP Tel: +44 (0) 207 594 1001 Fax : +44 (0) 207 594 1006 DNA sequencing An Osprey BSA system scan showing the denaturation of pH2, 5mg/ml Hen Egg White Lysozyme (HEWL). The scan shows the transmission of UV light through the protein sample along the temperature gradient. The absorption of the protein can be seen to decrease, giving increasing transmission, as the protein unfolds. A 3% increase is seen in transmission from native to denatured protein. The unfolding temperature of HEWL is measured as 53.8 ºC. This value is reproducible and matches that measured in a conventional UV spectrophotometer. The output spectrum of an UV LED emitting at 280 ± 8 nm is shown as observed directly on-axis by the spectrometer probe in the left hand picture. On the right, the off-axis fluorescence emission spectrum of a pure tryptophan amino acid sample illuminated by the same source is shown. The sharp peaks to the right of the broad fluorescence peak are caused by ambient light and can be subtracted. The Peregrine HCPE Analyzer UV absorption as a technique to detect and quantify biomolecules is routine in molecular biology. LFII™ is a quantum leap up from this state to one that allows the effective imaging of separating biomolecules in an electrophoretic system. The current Peregrine system is based on standard capillary electrophoresis (CE) of biomolecules such as DNA and proteins, small molecules and larger entities such as bacteria and viruses. The analytes are generated using standard off the shelf sample preparation kits, but without the addition of any label. Typically the amount of analyte injected is remarkably small, only a few 100 picolitres being normal. The sample is electrophoresed and separated as in any similar CE system. LFII™ is a combination of: - •The multi-point detection of molecules with no labels, dyes, stains or radioactivity •The use of advanced signal processing algorithms derived from particle and astrophysics •The imaging of biomolecules in capillary and microfluidic separation systems LFII™ is FAST, SENSITIVE AND REPRODUCIBLE. It has excellent QUANTIFICATION, is CFR21/11 compatible and delivers DIGITAL data. Peregrine High-Performance Capillary Electrophoresis (HPCE) Analyzer The Peregrine HPCE Protein and Nucleic Acid Analyzer provides fast, high quality label free separations of proteins, glycoproteins, peptides, bacteria viruses and small molecules. Based on a single-channel capillary system, it provides at least five times the resolution, excellent sensitivity, and superior reproducibility compared to competing systems, w ith precision digital quantification and low running costs. How do deltaDOT achieve Label Free Intrinsic Imaging? - the application of particle physics to molecular biology Standard capillary electrophoresis systems are limited to a single detection point. deltaDOT’s LFII TM technology uses multi-point detection of biomolecules as they are being separated in capillaries or microfluidic chip devices by standard electrophoresis. Multi-point detection allow s a connection betw een the position of the biomolecule as it traverses the detector, and the time it is detected, to be made. This space-time correlation is a pow erful and patented technique that regains the signal to noise lost w hen no labels are used. This correlation is analysed by signal processing algorithms draw from particle and astrophysics. Merlin High Resolution DNA Analysis - Sequencing - Short Tandem Repeat Osprey Biomolecule Stability Analyzer - Protein - Nucleic Acids LFII™ sequence data. The data shown here is just under 200 bases and was acquired in about 1 hour. The sequence was generated using a standard off-the shelf cyclic sequencing kit The sequence separations were carried out using detection at 254nm. Fused-silica capillaries 75µm internal diameter with a separation length of 70cm and total length 89cm were used. The different bases are identified by deltaDOT’s Virtual Colour technology There is a constant and growing need to sequence short templates of DNA. Genetic manipulations of single bases, such as point mutations, or larger DNA fragments, as in gene silencing; are now routine in many labs. SNP validation is also becoming increasingly important as their role in genetic disorders become clear. Further applications for short template sequencing are in Pharmacogenomics (i.e. which drug is right for which patient) and immunology (e.g. HLA typing). Other applications in Biodefence and forensics are also becoming important such as identification applications for a wide range of purposes such as disaster genetics. Sequencing is the best possible way to check these processes, and generally a template of less than 100 bases needs to be read. Merlin is designed to be high through in the sense of bench-top, on demand sequencing. No wait, no queue, no label. Short Tandem Repeat Analysis Short tandem Repeats or STR’s are short sequences of DNA that are repeated numerous times in a head-tail manner. They are usually found in non-coding regions of DNA surrounding the centromere (the structural center of the chromosomes). The variable number of repeated element for a given STR allele can distinguish different individuals in a population as they are unique to that individual. deltaDOT Virtual ID Data. Extra,potentially important stutter peaks are visible along with colour coded allele data Conventional STR dye defined data The Osprey) is a microfluidic chip-based instrument that applies aspects of LFII™ to the characterization of the properties of proteins, rather than to their separations. Osprey targets the market for QA/QC of biopharmaceuticals as its first application. Beyond the initial target application in biopharmaceutical QA/QC, deltaDOT is exploring broader applications in protein profiling, such as in drug discovery, protein engineering and other areas. deltaDOT Technology Label Free Intrinsic Imaging (LFII™) Peptide analysis by capillary zone electrophoresis (CZE) Detection of Bacteria and Viruses Peptide Standards MWs (Da): 1.Leucin enkephalin: 555 2.Bradykinin fragment: 572 3.Methionine enkephalin: 573 4.Bradykinin: 1059 5.Oxytocin: 1009 6.[Arg 8 ]- Vasopressin: 1087 7.Luteinising hormone releasing hormone: 1207 8.Substance P: 1347 9.Bombesin: 1638 ESI-Mass Spectrometry Detection of Ribonuclease B Glycoforms Peregrine High Resolution Analysis in R&D & QA/QC - Proteomics - DNA/RNA size analysis - Small molecule analysis - Bacteria/Viral Analysis The upper plot shows results from an ESI-MS. The lower trace 5 consecutive RNase B, generated using LFII™. Discrimination and signal-to-noise ratio in the LFII™ analysis are comparable to that of the more expensive and labor-intensive system, and the molecular weight determinations are highly accurate. LFII™ technology is suited to a wide variety of applications including the detection of non-tradition electrophoretic analytes. The top panel show the detection of whole E.coli . from a standard growth culture. The lower panel shows very early results of a dilution series of a Baculovirus culture, the black peak is 1:10 of stock (2.97X10 7 pfu/ml), red, 1:20, green 1:30 and blue, 1:40. The linearity of the peak height shows that this system has potential be replace plaque forming assays as a means of quantify virus titres. deltaDOT Peregrine The LFII TM data shows 9 consecutive CZE runs of SIGMA peptide standards Small Molecule Analysis The LFII TM data shows 14 consecutive CZE runs of the non-steroid based anti- inflammatory based drug, Ketoprofen
