RESUMEN
Design and successful implementation of a fully-integrated CMOS fluorescence biochip for DNA/RNA testing in molecular diagnostics (MDx) is presented. The biochip includes a 32×32 array of continuous wave fluorescence detection biosensing elements. Each biosensing element is capable of having unique DNA probe sequences, wavelength-selective multi-dielectric emission filter (OD of 3.6), resistive heater for thermal cycling, and a high performance and programmable photodetector. The dimension of each biosensor is 100µm×100µm with a 50µm×50µm Nwell-Psub photodiode acting as the optical transducer, and a ΣΔ modulator based photocurrent sensor. The measured photodetector performance shows ~116 dB detection dynamic range (10fA - 10nA) over the 25°C - 100°C temperature range, while being ~1 dB away from the fundamental shot-noise limit. To empirically demonstrate the compatibility of this biochip with MDx applications, we have successfully utilized the array and its thermal cycling capability to adopt a 7-plex panel for detection of 6 human upper respiratory viruses.
RESUMEN
A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virologic failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5' and 3' flanking sequences but having different centrally located nucleotides complementary to six codons at HIV-1 DRM reverse transcriptase position 103: AAA, AAC, AAG, AAT, AGA, and AGC. We then performed in vitro experiments using 80-mer oligonucleotides and PCR-amplified DNA from clinical plasma HIV-1 samples and culture supernatants that contained subtype A, B, C, D, CRF01_AE, and CRF02_AG viruses. Multiplexed solid-phase melt curve analysis discriminated perfectly among each of the six reported reverse transcriptase position 103 codons in both 80-mers and clinical samples. The sensitivity and specificity for detecting targets that contained AAC mixed with targets that contained AAA were >98% when AAC was present at a proportion of ≥10%. Multiplexed solid-phase melt curve analysis is a promising approach for developing point-of-care assays to distinguish between different codons in genetically variable regions such as those surrounding HIV-1 DRMs.
Asunto(s)
Farmacorresistencia Viral , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Pruebas en el Punto de Atención , Bases de Datos Genéticas , Genotipo , Infecciones por VIH/diagnóstico , Infecciones por VIH/tratamiento farmacológico , Humanos , Mutación , ARN ViralRESUMEN
PCR-based techniques are widely used to identify disease causing bacterial and viral pathogens, especially in point-of-care or near-patient clinical settings that require rapid results and sample-to-answer workflows. However, such techniques often fail to differentiate between closely related species that have highly variable genomes. Here, a homogenous (closed-tube) pathogen identification and classification method is described that combines PCR amplification, array-based amplicon sequence verification, and real-time detection using an inverse fluorescence fluorescence-resonance energy transfer technique. The amplification is designed to satisfy the inclusivity criteria and create ssDNA amplicons, bearing a nonradiating quencher moiety at the 5'-terminus, for all the related species. The array includes fluorescent-labeled probes which preferentially capture the variants of the amplicons and classify them through solid-phase thermal denaturing (melt curve) analysis. Systematic primer and probe design algorithms and empirical validation methods are presented and successfully applied to the challenging example of identification of, and differentiation between, closely related human rhinovirus and human enterovirus strains.
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The emergence of pathogens resistant to existing antimicrobial drugs is a growing worldwide health crisis that threatens a return to the pre-antibiotic era. To decrease the overuse of antibiotics, molecular diagnostics systems are needed that can rapidly identify pathogens in a clinical sample and determine the presence of mutations that confer drug resistance at the point of care. We developed a fully integrated, miniaturized semiconductor biochip and closed-tube detection chemistry that performs multiplex nucleic acid amplification and sequence analysis. The approach had a high dynamic range of quantification of microbial load and was able to perform comprehensive mutation analysis on up to 1,000 sequences or strands simultaneously in <2 h. We detected and quantified multiple DNA and RNA respiratory viruses in clinical samples with complete concordance to a commercially available test. We also identified 54 drug-resistance-associated mutations that were present in six genes of Mycobacterium tuberculosis, all of which were confirmed by next-generation sequencing.
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Virus ADN/efectos de los fármacos , Genotipo , Mycobacterium tuberculosis/efectos de los fármacos , Virus ARN/efectos de los fármacos , Semiconductores , Recuento de Colonia Microbiana , Sondas de ADN , Virus ADN/genética , Virus ADN/aislamiento & purificación , ADN Viral/análisis , Farmacorresistencia Bacteriana/genética , Farmacorresistencia Viral/genética , Estudios de Factibilidad , Genoma Bacteriano , Humanos , Miniaturización , Mutación , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Virus ARN/genética , Virus ARN/aislamiento & purificación , ARN Viral/análisisRESUMEN
We constructed a library of >10(12) unique, covalently coupled mRNA-protein molecules by randomizing three exposed loops of an immunoglobulin-like protein, the tenth fibronectin type III domain (10Fn3). The antibody mimics that bound TNF-alpha were isolated from the library using mRNA display. Ten rounds of selection produced 10Fn3 variants that bound TNF-alpha with dissociation constants (K(d)) between 1 and 24 nM. After affinity maturation, the lowest K(d) measured was 20 pM. Selected antibody mimics were shown to capture TNF-alpha when immobilized in a protein microarray. 10Fn3-based scaffold libraries and mRNA-display allow the isolation of high-affinity, specific antigen binding proteins; potential applications of such binding proteins include diagnostic protein microarrays and protein therapeutics.
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Anticuerpos/química , Evolución Molecular Dirigida/métodos , Fibronectinas/genética , Imitación Molecular , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Técnicas Químicas Combinatorias , Fibronectinas/química , Fibronectinas/metabolismo , Humanos , Datos de Secuencia Molecular , Análisis por Matrices de Proteínas , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A fluorescent Hoechst 33258 derivative has been prepared in which a hexa(ethylene glycol) linker is attached to the terminal phenol residue. Conjugation of this derivative to DNA sequences is accomplished by a reversed coupling protocol, one in which the 5'-terminal nucleoside residue of a fully protected DNA sequence is converted to a terminal phosphoramidite. In the presence of the Hoechst derivative and tetrazole the final coupling reaction is achieved to generate the conjugated nucleic acid. After deprotection and cleavage of the conjugate from the support, HPLC analysis indicates that the conjugation reaction proceeds with yields as high as 75%. The presence of the conjugated Hoechst derivative increases the stability of DNA duplexes typically by 10-16 degrees C. A variety of sequence variants indicate that the tether length is sufficient to reach beyond the terminus of the DNA duplex and bind to internal A-T rich target sequences as far away as four base pairs from the site of attachment. A four base pair binding site appears to be necessary for effective helix stabilization by the conjugate, but in some cases can include a G-C base pair, which is consistent with a previous X-ray diffraction study regarding the binding of Hoechst 33258 to duplex DNA. When A-T base pairs alternate with G-C base pairs, a small but discernible increase is T(m) is observed (3.6 degrees C), indicating that binding to this sequence still occurs, but not in the same manner as to A-T rich sequences. Upon formation of the conjugated duplex, an enhanced quantum yield for the fluorescence emission spectrum of the tethered Hoechst derivative is observed. When an A-T rich binding site is present, the enhanced quantum yield increases by at least 16- and in some cases to nearly 30-fold relative to the value obtained for the single-stranded DNA-Hoechst conjugate.
RESUMEN
Site-specific recombinases of the gamma Int family carry out two single-strand exchanges by binding as head-to-head dimers on inverted core-type DNA sites. Each protomer may cleave its own site as a monomer in cis (as for Cre recombinase), or it may recruit the tyrosine from its partner in trans to form a composite active site (as for Flp recombinase). The crystal structure of the gamma Int catalytic domain is compatible with both cleavage mechanisms, but two previous biochemical studies on gamma integrase (Int) generated data that were not in agreement. Support for cis and trans cleavage came from assays with bispecific DNA substrates for gamma and HK022 Ints and from functional complementation between recombination-deficient mutants, respectively. The data presented here do not provide new evidence for cis cleavage, but they strongly suggest that the previously described complementation results cannot be used in support of a trans-cleavage mechanism. We show here that IntR212Q retains some residual catalytic function but is impaired in binding to core-type DNA on linear substrates and in forming higher-order attL intasome structures. The binding-proficient mutant IntY342F can stabilize IntR212Q binding to core-type DNA through protein-protein interactions. Similarly, the formation of higher-order Int complexes with arm- and core-type DNA is boosted with both mutants present. This complementation precedes cleavage and thus precludes any conclusions about the mechanism of catalysis. Cross-core stimulation of wild-type HK022-Int cleavage on its cognate site (in cis) by mutant gamma Ints on bispecific core DNA suicide substrates is shown to be independent of the catalytic tyrosine but appears to be proportional to the respective core-binding affinities of the gamma Int mutants.
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Bacteriófago lambda/enzimología , ADN Viral/metabolismo , Integrasas/genética , Integrasas/metabolismo , Bacteriófago lambda/genética , Sitios de Unión , Prueba de Complementación Genética , Integrasas/química , Mutación , Oligonucleótidos/química , Oligonucleótidos/metabolismo , Recombinación GenéticaRESUMEN
An mRNA-protein fusion consists of a polypeptide covalently linked to its corresponding mRNA. These species, prepared individually or en masse by in vitro translation with a modified mRNA conjugate (the PROfusion process), link phenotype to genotype and enable powerful directed evolution schemes. We have exploited the informational content of the nucleic acid component of the mRNA-protein fusion to create an addressable protein microarray that self-assembles via hybridization to surface-bound DNA capture probes. The nucleic acid component not only directs the mRNA-protein fusion to the proper coordinate of the microarray, but also positions the protein in a uniform orientation. We demonstrate the feasibility of this protein chip concept with several mRNA-protein fusions, each possessing a unique peptide epitope sequence. These addressable proteins could be visualized on the microarray both by autoradiography and highly specific monoclonal antibody binding. The anchoring of the protein to the chip surface is surprisingly robust, and the system is sensitive enough to detect sub-attomole quantities of displayed protein without signal amplification. Such protein arrays should be useful for functional screening in massively parallel formats, as well as other applications involving immobilized peptides and proteins.