DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and in Vivo Applications.

Phage display links the phenotype of displayed polypeptides with the DNA sequence in the phage genome and offers a universal method for the discovery of proteins with novel properties. However, the display of large multisubunit proteins on phages remains a challenge. A majority of protein display systems are based on monovalent phagemid constructs, but methods for the robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼ 200 kDa tetrameric l-asparaginase protein on M13 and fd phages produced by ligation of SpyCatcher-Asparaginase fusion (ScA) and PEGylated-ScA (PEG-ScA) to barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 or p8 coat proteins yielded constructs with five copies of ScA displayed on p3 (ScA-p3), ∼100 copies of ScA on p8 protein (ScA-p8) and ∼300 copies of PEG-ScA on p8 protein (PEG-ScA-p8). Display constructs of different valencies and chemical modifications on protein (e.g., PEGylation) can be injected into mice and analyzed by deep sequencing of the DNA barcodes associated with phage clones. In these multiplexed studies, we observed a density and protein-dependent clearance rate in vivo. Our observations link the absence of PEGylation and increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of l-asparaginase on phages could be used to study the circulation life of this protein in vivo, and such an approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multisubunit proteins in vivo.

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water.

Flow cytomtery (FCM) has become a standard approach to enumerate viruses in water research. However, the nature of the fluorescent signal in flow cytometric analysis of water samples and the mechanism of its formation, have not been addressed for bacteriophages expected in wastewaters. Here we assess the behaviour of fluorescent DNA-staining dyes in aqueous solutions, as well as sensitivity and accuracy of FCM for enumeration of DNA-stained model bacteriophages λ, P1, and T4. We demonstrate that in aqueous systems fluorescent dyes form a self-stabilized (pseudolyophilic) emulsion of auto-fluorescing colloid particles. Sample shaking and addition of surfactants enhance auto-fluorescence due to increased dispersion and, in the presence of surfactants, stabilization of the dye emulsion. Bacteriophages with genome sizes <100 kbp (i.e. λ & P1) did not generate a distinct population signal to be detected by one of the most sensitive FCM instruments available (BD LSR Fortessa™ X-20), whereas the larger T4 bacteriophage was resolved as a distinct population of events. These results indicate that the use of fluorescent dyes for bacteriophage enumeration by flow cytometry can produce false positive signals and lead to wrong estimation of total virus counts by misreporting colloid particles as virions, depending on instrument sensitivity.

A lab-on-chip for malaria diagnosis and surveillance.

BACKGROUND: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges. METHODS: The platform consists of a disposable plastic chip and a low-cost, portable, real-time PCR machine. The chip contains a desiccated hydrogel with reagents needed for Plasmodium specific PCR. Chips can be stored at room temperature and used on demand by rehydrating the gel with unprocessed blood, avoiding the need for sample preparation. These chips were run on a custom-built instrument containing a Peltier element for thermal cycling and a laser/camera setup for amplicon detection. RESULTS: This diagnostic was capable of detecting all Plasmodium species with a limit of detection for Plasmodium falciparum of 2 parasites/µL of blood. This exceeds the sensitivity of microscopy, the current standard for diagnosis in the field, by ten to fifty-fold. In a blind panel of 188 patient samples from a hyper-endemic region of malaria transmission in Uganda, the diagnostic had high sensitivity (97.4%) and specificity (93.8%) versus conventional real-time PCR. The test also distinguished the two most prevalent malaria species in mixed infections, P. falciparum and Plasmodium vivax. A second blind panel of 38 patient samples was tested on a streamlined instrument with LED-based excitation, achieving a sensitivity of 96.7% and a specificity of 100%. CONCLUSIONS: These results describe the development of a lab-on-chip PCR diagnostic from initial concept to ready-for-manufacture design. This platform will be useful in front-line malaria diagnosis, elimination programmes, and clinical trials. Furthermore, test chips can be adapted to detect other pathogens for a differential diagnosis in the field. The flexibility, reliability, and robustness of this technology hold much promise for its use as a novel molecular diagnostic platform in developing countries.

A novel method for sample delivery and testing of whole blood: gel strip PCR for point of care (POC) molecular diagnostics.

Testing of whole blood in miniaturized PCR is compromised by the opaque nature of whole blood that leads to physical masking of a fluorescent signal. We demonstrate a method to perform real-time PCR with whole blood that avoids interference from the opacity of whole blood.

A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs.

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h. We here describe a multiparameter PCR assay to simultaneously detect HSV-1 and HSV-2 DNA templates, together with integrated positive and negative controls, with product detection by melting curve analysis (MCA), in an array of semi-solid polyacrylamide gel posts. Each gel post is 0.67 µL in volume, and polymerized with all the components required for PCR. Both PCR and MCA can currently be performed in one hour and 20 min. Unprocessed genital swabs collected in universal transport medium were directly added to the reagents before or after polymerization, diffusing from atop the gel posts. The gel post platform detects HSV templates in as little as 2.5 nL of raw sample. In this study, 45 genital swab specimens were tested blindly as a preliminary validation of this platform. The concordance of PCR on gel posts with conventional PCR was 91%. The primer sequestration method introduced here (wherein different primers are placed in different sets of posts) enables the simultaneous detection of multiple pathogens for the same sample, together with positive and negative controls, on a single chip. This platform accepts unprocessed samples and is readily adaptable to detection of multiple different pathogens or biomarkers for point-of-care diagnostics.

In-gel technology for PCR genotyping and pathogen detection.

This work describes the use of polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semisolid posts that serve as reaction vessels for parallel PCR amplification of an externally added template. DNA amplification occurred in a cylindrical, self-standing 9 × 9 array of gel posts each less than 1 µL in volume. Photopolymerization of the gel with an intercalating dye added prior to polymerization permitted acquisition of real-time PCR data and melting curve analysis data without the need for any type of post-PCR staining procedures. PCR was equally efficient and reproducible when template DNA was polymerized within the gel or when exogenous template was added atop precast gel posts. PCR amplification occurred with template from purified DNA or from raw urine of patients with BK viruria. Multiple primer sets can be utilized per gel post array with no detectable cross contamination. As few as 34 BK virus templates were consistently detected by PCR in an individual gel post. Amplification of HPA1 and FGFR2 genes in human genomic DNA (gDNA) required as little as 2-5 ng of gDNA template/gel post. The device prototype includes a Peltier element for PCR thermal cycling and a CCD camera to capture fluorescence for product detection. Our technology is amenable to integration in point of care microdevices.

Strategies for enhancing the speed and integration of microchip genetic amplification.

In this work, we explore the use of methods that allow a significant acceleration of genetic analysis within microchips fabricated from low thermal conductivity materials such as glass or polymers. Although these materials are highly suitable for integrating a number of genetic analysis techniques onto lab-on-a-chip devices, their low thermal conductivity limits the rate at which heat can be transferred and hence lowers the speed of thermal cycling. However, short thermal cycling times are the key to bringing PCR to clinical point-of-care applications. Although shrinking the PCR reaction chamber volume can increase the speed of thermal cycling, this strategy is not always suitable, particularly when dealing with clinical samples with low analyte concentrations. In the present work, we combine two alternate strategies for decreasing the time required to perform PCR: implementing a heat sink and optimizing the PCR protocol. First, the heat sink substantially reduces the thermal resistance opposing heat dissipation into the ambient environment, and eliminates the parasitic thermal capacitance of the regions in the microchip that do not require heating. The low thermal conductivity of glass is used to our advantage to design the heat-sink placement to achieve fast thermal transitions while maintaining low power consumption. Second, we explore the application of two-stage PCR to provide a further reduction in the time required to perform genetic amplification by merging the annealing and extension stages of the commonly used three-stage PCR approach. In combination, we reduce the time required to perform thermal cycling by roughly a factor of 3 while improving the temperature control.

Microfluidic platform for single nucleotide polymorphism genotyping of the thiopurine S-methyltransferase gene to evaluate risk for adverse drug events.

Prospective clinical pharmacogenetic testing of the thiopurine S-methyltransferase gene remains to be realized despite the large body of evidence demonstrating clinical benefit for the patient and cost effectiveness for health care systems. We describe an entirely microchip-based method to genotype for common single nucleotide polymorphisms in the thiopurine S-methyltransferase gene that lead to serious adverse drug reactions for patients undergoing thiopurine therapy. Restriction fragment length polymorphism and allele-specific polymerase chain reaction have been adapted to a microfluidic chip-based polymerase chain reaction and capillary electrophoresis platform to genotype the common *2, *3A, and *3C functional alleles. In total, 80 patients being treated with thiopurines were genotyped, with 100% concordance between microchip and conventional methods. This is the first report of single nucleotide polymorphism detection using portable instrumentation and represents a significant step toward miniaturized for personalized treatment and automated point-of-care testing.

Microfluidic chips for detecting the t(4;14) translocation and monitoring disease during treatment using reverse transcriptase-polymerase chain reaction analysis of IgH-MMSET hybrid transcripts.

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis. On microfluidic chips, we demonstrated accurate and versatile detection of molecular signatures in individual cancer cells, with value for monitoring response to therapy, detecting residual cancer cells that mediate relapse, and evaluating prognosis. Thus, testing for two clinically important molecular biomarkers, the IgH VDJ signature and hybrid transcripts signaling the t(4;14) chro-mosomal translocation, with predictive value in diagnosis, treatment decisions, and monitoring has been efficiently implemented on a miniaturized microfluidic system.

CD4 T cells play major effector role and CD8 T cells initiating role in spontaneous autoimmune myocarditis of HLA-DQ8 transgenic IAb knockout nonobese diabetic mice.

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab(0); DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown. In the present study, we first investigated whether NOD/Ab(0) mice transgenic for a different DQ allele, DQ6, (DQA1*0102, DQB1*0602) would also develop myocarditis. NOD.DQ6/Ab(0) animals showed no cardiac pathology, implying that DQ8 is specifically required for the myocarditis phenotype. To further characterize the cellular immune mechanisms, we established crosses of our NOD.DQ8/Ab(0) animals with Rag1 knockout (Rag1(0)), Ig H chain knockout (IgH(0)), and beta(2)-microglobulin knockout (beta(2)m(0)) lines. Adoptive transfer of purified CD4 T cells from NOD.DQ8/Ab(0) mice with complete heart block (an indication of advanced myocarditis) into younger NOD.DQ8/Ab(0) Rag1(0) animals induced cardiac pathology in all recipients, whereas adoptive transfer of purified CD8 T cells or B lymphocytes had no effect. Despite the absence of B lymphocytes, NOD.DQ8/Ab(0)IgH(0) animals still developed complete heart block, whereas NOD.DQ8/Ab(0)beta(2)m(0) mice (which lack CD8 T cells) failed to develop any cardiac pathology. CD8 T cells (and possibly NK cells) seem to be necessary to initiate disease, whereas once initiated, CD4 T cells alone can orchestrate the cardiac pathology, likely through their capacity to recruit and activate macrophages. Understanding the cellular immune mechanisms causing spontaneous myocarditis/IDCM in this relevant animal model will facilitate the development and testing of new therapies for this devastating disease.

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history.

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods. To address this issue, we are employing a bacterial artificial chromosome (BAC) cloning strategy to sequence entire MHC haplotypes from consanguineous cell lines as part of the MHC Haplotype Project. Here we present 4.25 Mb of the human haplotype QBL (HLA-A26-B18-Cw5-DR3-DQ2) and compare it with the MHC reference haplotype and with a second haplotype, COX (HLA-A1-B8-Cw7-DR3-DQ2), that shares the same HLA-DRB1, -DQA1, and -DQB1 alleles. We have defined the complete gene, splice variant, and sequence variation contents of all three haplotypes, comprising over 259 annotated loci and over 20,000 single nucleotide polymorphisms (SNPs). Certain coding sequences vary significantly between different haplotypes, making them candidates for functional and disease-association studies. Analysis of the two DR3 haplotypes allowed delineation of the shared sequence between two HLA class II-related haplotypes differing in disease associations and the identification of at least one of the sites that mediated the original recombination event. The levels of variation across the MHC were similar to those seen for other HLA-disparate haplotypes, except for a 158-kb segment that contained the HLA-DRB1, -DQA1, and -DQB1 genes and showed very limited polymorphism compatible with identity-by-descent and relatively recent common ancestry (<3,400 generations). These results indicate that the differential disease associations of these two DR3 haplotypes are due to sequence variation outside this central 158-kb segment, and that shuffling of ancestral blocks via recombination is a potential mechanism whereby certain DR-DQ allelic combinations, which presumably have favoured immunological functions, can spread across haplotypes and populations.

Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.

Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma. High abundance IgH VDJ transcripts amplified in conventional systems or by capillary electrophoresis through channels on microfluidic chips or, alternatively, PCR product amplified from individual myeloma plasma cells in a single stage RT-PCR reaction was readily detectable on microfluidic chips. For low abundance templates, a nested PCR strategy was needed to detect PCR product by any method. Using microfluidic chips, PCR products amplified from genomic IgH VDJ DNA were detected in six out of eight plasma cells. Comparison of the ABI3100 and the microfluidic chip indicates that approximately 20 times more sample is injected into the ABI 3100 capillary than for the microfluidics chip. Overall, for high and low abundance template in individual cells, the microfluidic separation/detection system is at least as sensitive as the ABI 3100. In the future, integrated microfluidic platforms that incorporate both PCR cycling and product detection on the same chip are likely to exceed conventional systems in sensitivity and speed of genetic analysis by RT-PCR or PCR.

Complete MHC haplotype sequencing for common disease gene mapping.

The future systematic mapping of variants that confer susceptibility to common diseases requires the construction of a fully informative polymorphism map. Ideally, every base pair of the genome would be sequenced in many individuals. Here, we report 4.75 Mb of contiguous sequence for each of two common haplotypes of the major histocompatibility complex (MHC), to which susceptibility to >100 diseases has been mapped. The autoimmune disease-associated-haplotypes HLA-A3-B7-Cw7-DR15 and HLA-A1-B8-Cw7-DR3 were sequenced in their entirety through a bacterial artificial chromosome (BAC) cloning strategy using the consanguineous cell lines PGF and COX, respectively. The two sequences were annotated to encompass all described splice variants of expressed genes. We defined the complete variation content of the two haplotypes, revealing >18,000 variations between them. Average SNP densities ranged from less than one SNP per kilobase to >60. Acquisition of complete and accurate sequence data over polymorphic regions such as the MHC from large-insert cloned DNA provides a definitive resource for the construction of informative genetic maps, and avoids the limitation of chromosome regions that are refractory to PCR amplification.