Theoretical Design and Analysis of Multivolume Digital Assays with Wide Dynamic Range Validated Experimentally with Microfluidic Digital PCR

This paper presents a protocol using theoretical methods and free software to design and analyze multivolume digital PCR (MV digital PCR) devices; the theory and software are also applicable to design and analysis of dilution series in digital PCR. MV digital PCR minimizes the total number of wells required for “digital” (single molecule) measurements while maintaining high dynamic range and high resolution. In some examples, multivolume designs with fewer than 200 total wells are predicted to provide dynamic range with 5-fold resolution similar to that of single-volume designs requiring 12 000 wells. Mathematical techniques were utilized and expanded to maximize the information obtained from each experiment and to quantify performance of devices and were experimentally validated using the SlipChip platform. MV digital PCR was demonstrated to perform reliably, and results from wells of different volumes agreed with one another. No artifacts due to different surface-to-volume ratios were observed, and single molecule amplification in volumes ranging from 1 to 125 nL was self-consistent. The device presented here was designed to meet the testing requirements for measuring clinically relevant levels of HIV viral load at the point-of-care (in plasma, 1 000 000 molecules/mL), and the predicted resolution and dynamic range was experimentally validated using a control sequence of DNA. This approach simplifies digital PCR experiments, saves space, and thus enables multiplexing using separate areas for each sample on one chip, and facilitates the development of new high-performance diagnostic tools for resource-limited applications. The theory and software presented here are general and are applicable to designing and analyzing other digital analytical platforms including digital immunoassays and digital bacterial analysis. It is not limited to SlipChip and could also be useful for the design of systems on platforms including valve-based and droplet-based platforms. In a separate publication by Shen et al. (J. Am. Chem. Soc., 2011, DOI: 10.1021/ja2060116), this approach is used to design and test digital RT-PCR devices for quantifying RNA

INDUCTION OF TOLERANCE BY ORAL ADMINISTRATION OF FACTOR VIII AND TREATMENT OF HEMOPHILA

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a Subject a therapeutically effective amount of the deficient anti gen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac atherapeutically effective amount of the appropriate clotting factor other than in a liposome, Sufficient to induce oral tolerance and Supply exogenous clotting factor to the subject

Searching the World-Wide-Web using nucleotide and peptide sequences

*Background:* No approaches have yet been developed to allow instant searching of the World-Wide-Web by just entering a string of sequence data. Though general search engines can be tuned to accept ‘processed’ queries, the burden of preparing such ‘search strings’ simply defeats the purpose of quickly locating highly relevant information. Unlike ‘sequence similarity’ searches that employ dedicated algorithms (like BLAST) to compare an input sequence from defined databases, a direct ‘sequence based’ search simply locates quick and relevant information about a blunt piece of nucleotide or peptide sequence. This approach is particularly invaluable to all biomedical researchers who would often like to enter a sequence and quickly locate any pertinent information before proceeding to carry out detailed sequence alignment. 

*Results:* Here, we describe the theory and implementation of a web-based front-end for a search engine, like Google, which accepts sequence fragments and interactively retrieves a collection of highly relevant links and documents, in real-time. e.g. flat files like patent records, privately hosted sequence documents and regular databases. 

*Conclusions:* The importance of this simple yet highly relevant tool will be evident when with a little bit of tweaking, the tool can be engineered to carry out searches on all kinds of hosted documents in the World-Wide-Web.

*Availability:* Instaseq is free web based service that can be accessed by visiting the following hyperlink on the WWW
http://instaseq.georgetown.edu 
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ORAL TREATMENT OF HEMOPHILIA

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a subject a therapeutically effective amount of the deficient antigen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac a therapeutically effective amount of the appropriate clotting factor other than in a liposome, sufficient to induce oral tolerance and supply exogenous clotting factor to the subject

Developments in the tools and methodologies of synthetic biology.

Synthetic biology is principally concerned with the rational design and engineering of biologically based parts, devices, or systems. However, biological systems are generally complex and unpredictable, and are therefore, intrinsically difficult to engineer. In order to address these fundamental challenges, synthetic biology is aiming to unify a body of knowledge from several foundational scientific fields, within the context of a set of engineering principles. This shift in perspective is enabling synthetic biologists to address complexity, such that robust biological systems can be designed, assembled, and tested as part of a biological design cycle. The design cycle takes a forward-design approach in which a biological system is specified, modeled, analyzed, assembled, and its functionality tested. At each stage of the design cycle, an expanding repertoire of tools is being developed. In this review, we highlight several of these tools in terms of their applications and benefits to the synthetic biology community

PRT: Parallel program for a full backtranslation of oligopeptides

DNA hybridization methods have become the most widely used tools in molecular biology to identify organisms and evaluate gene expression levels. PCR (Polymerase Chain Reaction)-based methods, fluorescent in situ hybridization (FISH) and the recent development of DNA microarrays as a high throughput technology need efficient primers or probes design. Evaluation of the metabolic capacities of complex microbial communities found in terrestrial or aquatic environments requires new probe design algorithms that reflect the genetic diversity. As only a small part of the microbial diversity is known, gene sequences deposited in international databases do not reflect the entire diversity. In this context we propose to use oligopeptide sequences for the design of complete set of DNA probes that are able to target the entire genetic diversity of genes encoding enzymes. Due to the degenerated genetic code backtranslation must be managed efficiently. To our knowledge no software has been developed to propose a full backtranslation. This complexity is tractable since we only need to focus on short oligopeptides for DNA probe design. We propose new algorithms that perform a high performance oligopeptide backtranslation into all potential nucleic sequences. We use different efficient techniques such as memory mapping to perform such a computing. We also propose a MPI parallel computing that reduces the whole execution time using data load balancing and network file stream distribution on a cluster architecture

Structure, stability and elasticity of DNA nanotube

DNA nanotubes are tubular structures composed of DNA crossover molecules. We present a bottom up approach for construction and characterization of these structures. Various possible topologies of nanotubes are constructed such as 6-helix, 8-helix and tri-tubes with different sequences and lengths. We have used fully atomistic molecular dynamics simulations to study the structure, stability and elasticity of these structures. Several nanosecond long MD simulations give the microscopic details about DNA nanotubes. Based on the structural analysis of simulation data, we show that 6-helix nanotubes are stable and maintain their tubular structure; while 8-helix nanotubes are flattened to stabilize themselves. We also comment on the sequence dependence and effect of overhangs. These structures are approximately four times more rigid having stretch modulus of ~4000 pN compared to the stretch modulus of 1000 pN of DNA double helix molecule of same length and sequence. The stretch moduli of these nanotubes are also three times larger than those of PX/JX crossover DNA molecules which have stretch modulus in the range of 1500-2000 pN. The calculated persistence length is in the range of few microns which is close to the reported experimental results on certain class of the DNA nanotubes.Comment: Published in Physical Chemistry Chemical Physic

A Data Transformation System for Biological Data Sources

Scientific data of importance to biologists in the Human Genome Project resides not only in conventional databases, but in structured files maintained in a number of different formats (e.g. ASN.1 and ACE) as well a.s sequence analysis packages (e.g. BLAST and FASTA). These formats and packages contain a number of data types not found in conventional databases, such as lists and variants, and may be deeply nested. We present in this paper techniques for querying and transforming such data, and illustrate their use in a prototype system developed in conjunction with the Human Genome Center for Chromosome 22. We also describe optimizations performed by the system, a crucial issue for bulk data