Targeted NGS for investigating the mitochondrial genome and heteroplasmy
Disrupted mitochondrial functions and genetic variants of mitochondrial DNA (mtDNA) have been observed in different human neoplasms. Next-generation sequencing (NGS) can be used to detect even low heteroplasmy-level mtDNA variants.…
LC Sciences is a global biotechnology company providing products and services to genomics and proteomics researchers across an array of markets for nucleic acid/protein analysis, biomarker-discovery and drug development.
Diverse ScFv library by Synthetic CDR Fragments led to Improvement of ERBB2 Targeting
Despite of the therapeutic promise, many of the present cancer therapeutic antibodies failed to enter the drug pipeline due to their lacking of specificity and efficiency which would require improved target binding affinity. We present herein an integrated method of antibody library construction and antibody maturation to accelerate the development of therapeutic antibody against specific cancer antigens. We demonstrate that a comprehensive large CDR fragment synthetic library of millions of predetermined sequences can be constructed using array chip technology to give useful materials, quickly and inexpensively for generation of humanized antibodies for phage display screening of cancer antigen targeting. ChA21 is an engineered single chain chimeric antibody targeting ErbB2 generated previously by our laboratory. Our crystal structure indicates that ChA21 recognize ERBB2 but at a different site compared to herceptain. Given the significant therapeutic benefit of Herceptain, which targets ERBB2 and is a marketed anticancer therapeutic antibody, our goal is to explore the therapeutic potential of ChA21 as an alternate/adjuvant or a more effective therapeutic antibody. In this presentation, we report the construction of millions of ChA21 variants using synthetic CDR fragments, which were designed based on our analysis of the crystal structure of the antibody-antigen interactions. The designed CDR fragments systematically covered single, double or triple mutations at the specific sites of each of the five CDRs (except for variable light chain CDR2) to give non biased diverse spectrum for screening of antigen binding (in millions of antibody variants). We used NGS sequencing to observe that the sequence diversity of the synthetic CDR library is as designed, and for rapidly extraction of phage display screening results, which provided information for us to build a second generation of CDR library for antibody variants to drive the screening of high affinity ERBB2 binding antibodies. Using the method, we achieved sub-nM binding, i.e, 10~30-fold binding affinity improvement for the ChA21 family of antibodies compared to herceptain binding to ERBB2. We further demonstrate that the high affinity binding antibody more effectively inhibit the growth of breast and gastric cancer cells.
Precise Control of Diversity for Synthetic Antibody Library Design & Construction
Synthetic antibody libraries have proven to be effective tools for drug discovery and development through the generation of functional, high-affinity antibodies against a wide variety of antigens. They are an expanding alternative to standard hybridoma technology especially for application to particularly difficult therapeutic challenges that cannot be addressed with antibodies from the natural repertoire. The performance of a synthetic antibody library depends in large part on the diversity of the library which must be designed based on thorough understanding of the antibody structure and function. Focused diversity can provide an efficient path to antibody candidates designed for exceptional performance in specialized applications if precise control over design and construction is exercised. The use of degenerate oligos and other standard methods of diversity introduction lack this precise control and can introduce unwanted or useless codons into the library, thus limiting its performance. Fully designed library diversity is enabled through parallel in-situ (on-chip) on synthesis of tens of thousands of specific (non-degenerate) oligonucleotide sequences We demonstrate the bioinformatics-based design and high-throughput synthesis of a mutant phage display library to improve affinity of anti-ErbB2 single chain monoclonal antibody A21
Breakthrough microRNA Discoveries
Study of microRNA in Endocrinology
What are miRNA’s functions in endocrinology? There is an intricate reciprocal relationship between these two important regulatory systems.
• Many miRNAs regulate hormones and many miRNAs are in turn regulated by hormones
• miRNAs have been shown to target many genes important for proper endocrine function and metabolism.
• Dysregulation of miRNAs can contribute to endocrine related diseases: Hormone dependent Cancers, Obesity, Diabetes, Hyperglycemia Lipodystrophy
microRNAs in Molecular Medicine
Molecular Diagnostics / Biomarkers – Identification of specific miRNAs or miRNA expression based signatures that can act as biomarkers for various diseases/pathologies.
1. Make accurate and detailed clinical diagnosis
2. Determine prognosis and predict treatment efficacy
3. Monitor and assess the health effects of environmental and other toxicants
Drug Discovery / Therapeutics – Identification of miRNAs that play essential roles in disease to act as drugs or possible therapeutic targets inhibitors.
1. miRNAs as tumor suppressor drugs
2. miRNAs as drug targets
3. Study of miRNAs to understand chemo/radio resistance
A Simple Multiplex PCR Approach for Target Enrichment in Next-Gen Sequencing
Multiplexing PCR is a simple way to extract genomic regions of interest for various genetic tests such as variant analysis or genotyping. Somatic mutations such as SNPs are unlikely to be best detected using regular whole genome sequencing and genotyping by sequencing (GBS) in any large genome species requires reduction of genome complexity. Therefore, many current genetic test workflows start with multiplexing PCR to extract genetic marker carrying regions from whole genomes before running hybridization, sequencing, or electrophoresis tests to identify the markers. We have developed a new multiplexing approach with a significantly simplified workflow and significantly improved robustness. When applied to sequencing target enrichment application, the workflow for producing amplified targets involves only one hands-on step and one PCR run. Sequencing library adapters, sample barcodes and molecular tags are all incorporated during this single PCR run. The approach is designed to require low sample input and to produce superior amplicon uniformity and sequence specificity. The approach involves a novel primer design and a proprietary reaction composition A PCR run consists of two functionally separated reaction phases namely target capture and library composition. phases, www.lcsciences.amplification, without any hands-on step in between.
Development of µPepArray as a Powerful Molecular Tool for Proteomic Profiling of Cellular Signaling Proteins (CSPs) of Pancreatic Cancer – Interrogate CSP Variations Induced by the Treatment of Tyrosine Kinase Inhibitors (TKIs)
It is well accepted that molecular profiling of cellular proteins or nucleic acids can offer answers to courses of diseases and underlined connections of pathogenic molecules, and thus, pointing out therapeutic targets. However, protein profiling, especially at the cellular level of protein profiling has been challenging. There is only limited choices to allow systematic investigation of cellular protein activities, such as their responses, i.e., sensitive, responsive or nonresponsive or resistant, to therapeutic treatment. This presentation reports peptide microarray chip (PepArray) technology developed as a powerful molecular tool for proteomic profiling of Cellular Signaling Proteins (CSPs) to interrogate CSP variations in pancreatic cancer induced by treatment of a new generation of anti-cancer therapies, i.e., tyrosine kinase therapeutics (TKls). PepArray chips encodes receptor kinase protein (RTK) interactions with phosphotyrosine (pY) motif binding domain proteins, revealing signaling network activities, which are translated into clinic relevant information valuable for therapeutic treatment. Examples of cellular protein profiling of pancreatic cancer treated with three generations of small molecule tyrosine kinase (EGFR) inhibitors (TKis): Erlotinib (TarcevaTM); Afatinib (Gilotrif TM); and the 2016 FDA approved AZD9291 (TagrissoTM) will be demonstrated. Our PepArrayTMs tudies revealed protein profiles as molecular signatures of the cellular conditions through proteins of commonly or differentially expressed. The Proteomic profiles of pancreatic cancer cells revealed 80 signaling proteins implicated in 39 cancer related pathways under Erlotinib treatment; while 135 signaling proteins implicated in 38 cancer related pathways under Afatinib treatment; and 78 signaling proteins associated with 39 cancer related pathways under AZD9291 treatment. Such information about functional cellular proteins provided valuable molecular signature, which reflect cancer status to allow assessment of effectiveness of cancer treatment, i.e., sensitive vs insensitive, responsive vs resistant. PepArray proteomic and signaling pathway results thus hold clinical significance in identifying molecular markers in therapeutic treatment of pancreatic cancer, and the various cancers, and in the cancer therapeutic strategy including monitoring and predicting of therapeutic effectiveness, will lead to a strong molecular basis for application of precision medicine to greatly benefit human health and wellness.
PepArray™ – Epitope Mapping on a Flexible High-Density Microfluidic Chip
Although a number of proteomics technologies are well-developed, the demand for reliable, sensitive, accurate, and comprehensive measurements of epitope binding remains unfulfilled. Through the use of overlapping peptides as epitopes on a custom synthesized addressable peptide microarray (PepArray™), we can systematically screen thousands of epitope sequences in a single experiment. A proprietary microarray platform and advanced microfluidic technologies ensure quantitative measurements of binding events. This combination of high-throughput capacity with quantitative measurement enables us to quickly and efficiently identify high affinity and high specificity target binding compounds. We have a novel, in situ, on-microchip synthesis technology (mParaflo®) that allows us to perform in situ synthesis of high-density peptide arrays according to custom designs. Forty-one 96-well titer plate experiments (i.e. conventional experiments) can be accomplished simultaneously on one chip. We also apply this flexible peptide array technology for rapid and reliable quantitative measurements for phosphorylation and protein binding. Our ultimate goal is to provide new, powerful research and clinical study tools to address the critical needs in proteomic research, clinical diagnosis, and therapeutic treatment.
Proteomic Kinase Substrate & Phosphopeptide Chip Technology for Protein Profiling
Here we present an advanced microfluidics microchip technology developed for systematic high-throughput screening of protein interactions. A novel chemistry enables custom synthesis of peptides and peptidomimetics directly on the high density microfluidic chip at addressable chip locations. Chip microfluidics provide a controlled, enclosed environment to conduct 4K-30K simultaneous protein binding reactions on a single chip. We will further present an application example demonstrating use of this peptide array technology to characterize and identify novel protein interactions from cancer signaling pathways. The interaction between phosphopeptides (PPEPs) and phosphoprotein binding domain containing proteins (PPBDs) on a microchip reveal novel protein cascades representing various signaling pathways through target binding.
Phosphopeptide Array Platform for Cellular Signaling Network Protein Profiling in Breast Cancer Cells
Tyrosine phosphorylation is the hallmark of activation of Receptor Tyrosine Kinase (RTK) pathway proteins which regulate various aspects of cellular lysis,Immunoprecipitation Cells division, multiplication, differentiation and apoptosis (1-2). Temporal and 1-containing 20mM Tris (pH 8) spatial misregulation of RTKs leads to various cancers (3). Phospho-protein enrichment coupled with high-throughput mass spectrometry methods have lead to cataloguing of thousands of such tyrosine phospho modifications on proteins and is still expanding rapidly (4-6). But understanding the functional significance of these phospho-motifs proteins in modulating signals that directs cells to attain the abnormal cancerous state and metastasize is still an enigma. Comprehensive maps of protein networks regulated by such phospho motifs will lead to identification of nodal signaling protein motifs and open up avenues for better therapeutic intervention strategies. Using a set of breast cancer cells, we have generated a detailed map of interaction between phospho-motifs representing various RTK pathway proteins and an RTK adaptor protein GRB2. We also describe a novel high throughput tyrosine phosphopetide microarray platform (μParaflo® PepArray Microchip system) and its potential applications in biomarker and drug discovery. We have uncovered several novel interactions of tyrosine phospho-motifs with GRB2 in breast cancer cell systems.
microRNA in the Cardiovascular System
Why Study miRNA in the Cardiovascular System?
1. Several miRNA genes are specifically expressed or highly enriched in skeletal and/or cardiac muscle, the so‐called muscle miRNAs.
2. miRNAs are essential for proper muscle development and exert post‐transcriptional control during myogenesis
3. Muscle miRNAs regulate myoblast proliferation and differentiation in skeletal muscle.
4. Circulating miRNAs may be novel biomarkers for coronary artery disease (CAD) and acute myocardial infarction (AMI)
5. Dysregulation of miRNAs can contribute to cardiovascular targets. related diseases and disorders: CAD, AMI, arrhythmia, hypertrophy and fibrosis.
6. miRNAs are likely associated with other muscle‐related diseases and are potential therapeutic targets
Genomics Discovery Applications for Microarray Synthesized Oligonucleotide Pools
Synthetic oligonucleotides (oligos) have proven to be effective life science tools for a wide range of applications, from PCR to sequencing. Traditionally, oligo synthesis of individual sequences is performed on a support (typically controlled pore glass [CPG]) packed into a synthesis “column” in varying quantities depending on yield requirements. The wide utility of oligos has driven the development of more scalable technologies to enable synthesis of many sequences in parallel. New applications such as target capture for next-gen sequencing are pushing this need even farther, to tens of thousands of sequences. Microarrays have proven a suitable method for parallel oligo synthesis; however, adapting the synthesis chemistry to massive parallel reactions on a solid surface has been challenging. Variations to traditional DMT protection of synthesis monomers such as electrochemical of photolabile protecting groups have had success but lack the reaction efficiency and flexibility to incorporate modified monomers. We have developed a microarray synthesis technology based on photogenerated acid (PGA) deprotection of standard DMT protected monomers which preserves both the high coupling efficiency and flexibility of traditional solid support oligo synthesis, but enables the massive parallel manufacture of tens of thousands of oligos sequences required for advanced applications. Oligos are synthesized on the microarray chip and then cleaved into solution, ready for use in multiplexing reactions. The cleaved oligos are identical to and can be modified in any way that oligos traditionally synthesized oligos can. Additionally, the ability to incorporate modified terminators, bases or backbone greatly increases the range of application of these oligos. Microarray synthesized oligos are now used in varying multiplex applications such as target capture for next-gen sequencing, gene synthesis and production of protein coding libraries.
Study of microRNA in Neuroscience – Featuring the Research of Dr. Walter J Lukiw – Tenured Professor of Neuroscience and Ophthalmology
Dr. WJ Lukiw’s major research interests are in small non‐coding RNAs (sncRNAs), such as micro RNAs (miRNAs), and messenger RNA (mRNA) complexity and speciation in the human brain during development and aging, and in the molecular-genetics, epigenetics and elucidation of inflammatory signaling circuits in human prion disease, in Alzheimer’s disease (AD), and in age-related macular degeneration (AMD). His laboratory also has a strong research interest in HSV-1 and viral infection of the central nervous system (CNS) and how it may contribute to inflammatory neurodegeneration.
Dr. Lukiw hypothesizes that specific pathways of genetic mis-regulation involving altered sncRNA miRNA and mRNA complexity and expression, in human brain and retinal cells lead to an inflammatory response resulting in apoptotic changes that are direct precursors to early pathological change in both AD and AMD.
Phospho-PepArray based Identification of Novel Protein Interaction Networks in Tyrosine Kinase Signaling Pathways
Protein phosphorylation mediates many critical cellular responses and is essential for many biological functions during development. About one-third of cellular proteins are phosphorylated, representing the phosphor-proteome, and phosphorylation can alter a protein’s function, activity, localization and stability. Tyrosine phosphorylation events mediated by aberrant activation of Receptor Tyrosine Kinase (RTK) pathways have been proven to be involved in the development of several diseases including cancer. With the available phosphorylation data on various High throughput proteomic technology platforms, it is becoming increasingly evident that many proteins are interlinked with each other in multiple signaling pathways through multiple protein phosphorylation sites (pT, pS and pY). Hence it is becoming extremely hard to target a specific protein as a biomarker or for a drug. Post –translational modifications (eg., pY) on proteins create multiple subgroups of the same proteins which are pathway specific. Hence a sensible strategy is to target these phosphorylated sites that are pathway specific. Here we present our microchip based (PepArrayTM) peptide microarray technology to characterize and identify novel protein interactions from cancer signaling pathways. The interaction between phosphopeptides (PPEPs) and phosphoprotein binding domain containing proteins (PPBDs) on a microchip reveal novel protein cascades representing various signaling pathways through target binding. More than 10 families of PPBDs (SH2, PTB, WW, 14-3- etc) represent a vast majority of proteins in a signaling cascade. Proteins with phosphotyrosine modifications not only bind to proteins with SH2 domains but also activate downstream proteins (Kinases and phosphatases) to initiate a signaling cascade. We illustrate Phosphotyrosine (pY) peptide interactions with an SH2 domain containing protein, GRB2 to identify protein complexes under various signaling cascades. We have used Breast cancer cells (T47D) and Human Umbilical vein cells (HUVEC) to demonstrate the differential signaling cascade mediated by differential protein-protein interaction networks. Further focus will be on technology advancement through integration of high density peptide microarray with computational web tools to rapidly identify novel protein interaction networks in various cancers and neurodegenerative diseases and increase the sensitivity of detecting protein interactions.
Site specific profiling of histone methyltransferases in cancer cells using histone peptide microarray containing a comprehensive set of histone peptides
Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Post-translational modifications (PTMs) of histones play a critical role in diverse biological processes including chromatin compaction, gene expression and cell differentiation. Among a myriad of PMTs, histone methylation catalyzed by histone methyltransferases (HMTs) has been increasingly recognized as an important player responsible for a major signaling mechanism in eukaryotic cells. This suite of epigenetic modifiers represents a new and promising class of therapeutic targets. In cancer, there is a growing body of evidence that suggests changes in the activity of HMTs (a class of chromatin-modifying enzymes) contribute to the uncontrolled cell proliferation that is a hallmark of this devastating disease. The sequence specificity of the substrates of HMTs under a cellular condition are largely unknown but known targets have been mostly identified through a conventional candidate-based approach by using purified HMTs. However, such an experiment frequently does not reflect what could be occurring in cellular contexts or in vivo. In this study, we designed and synthesized a comprehensive histone peptide microarray (PepArray) on a microfluidic chip which contains 3,919 peptides. The peptides contain nine residues with the methylation sites and mutant sites situated in the middle of the sequence so that each peptide has a unique possibility for modification such as methylation or acetylation. We obtained nuclear extract from the breast cancer cell line T47D, and applied the protein lysates to the histone methylation PepArray chip. After incubation of the chip with a methyl-specific antibody, significant signals were detected at the sites containing peptides corresponding to H2AK74, H3K122, and H4K59. We found null signal at mutant sites where the target lysine(K) was replaced with alanine(A). These results reveal the specific activity profiles of HMTs at defined histone sites in a cellular system. Planned further investigation will compare the different histone methylation or acetylation profiles in the various cellular systems, especially in different cancer systems. The current experiment demonstrates an effective solution to comprehensive studies of epigenetic modification. This information may be translated into therapeutic targets of histone methylation inhibition by focusing on identifying inhibitors of specific HMTs as targets for a new generation of therapeutics. Citation Format: Bing Zhu, Ailing Hong, Chris Hebel, Xiaochuan Zhou, Xiaolian Gao. Site specific profiling of histone methyltransferases in cancer cells using histone peptide microarray containing a comprehensive set of histone peptides.
Abstract 4235: Site specific profiling of histone…. Available from: https://www.researchgate.net/publication/273881009_Abstract_4235_Site_specific_profiling_of_histone_methyltransferases_in_cancer_cells_using_histone_peptide_microarray_containing_a_comprehensive_set_of_histone_peptides [accessed Apr 05 2018].
microRNAs in Toxicogenomics Selected Publications by LC Sciences’ Customers
What are miRNA’s functions in toxicogenomics?
1. miRNAs are effectors of environmental influences on gene expression. Thus miRNAs play an important role in the cellular response to toxicants and disease.
2. The expression of miRNAs, like many of the genes important in toxicology, can be regulated by xenobiotics and DNA methylation.
3. Xenobiotic‐mediated miRNA expression has been directly linked with downstream role in protein expression and cell proliferation.
From initial broad search to focused biological insights: An efficient microRNA discovery and profiling strategy.
Deep sequencing of RNA (RNA‐Seq) is a powerful new technology that yields results broadly covering genome‐wide microRNAs (miRNAs) from samples of various temporal and special Sequencing origins While the time tested approach microarray continues to be an effective tool to systematically profile and compare gene expression rapidly reproducibly and cost effectively Here we present a unique combination of the latest deep sequencing technology advanced bioinformatics and an innovative microfluidic custom microarray platform that leverages all these technologies Seq‐ArraySM is a customized synergistic solution to high‐throughput genome‐wide miRNA discovery and profiling for revealing regulatory target genes defining gene expression Sensitive pathways and discovering biomarkers The potential of this method to capture comprehensive sequencing information and systematically profile large groups of samples based on those sequencing findings is illustrated by an application example: A group of previously unknown small Novel RNAs was discovered and found to show significant differential expression in test conditions
Study of microRNA in Plants Recent published work by LC Sciences’ Customers
Basic Research / Discovery ‐ Identification of novel miRNAs in various plant species specific tissues and understanding their mechanism action and regulatory roles.
Stress Response – Identification of specific miRNA based markers that play essential roles in, plant growth, development, and stress response.
Plant Breeding – Identification of miRNAs that regulate key traits such as hybrid vigor, reproduction, could be useful for plant breeding and environmental protection programs; Germplasm screening – identification of miRNA based signatures for cataloguing plant genotypes and accessions.
Degradome Sequencing For Plant microRNA Target Identification
MicroRNAs (miRNAs) are endogenous small noncoding RNAs that play crucial roles in the post transcriptional regulation of gene expression in plants and animals. They function by binding to complementary mRNA molecules targets) and acting as negative regulators of translation. This function is part of a complex web as a single miRNA might have several target genes and a single gene may be regulated by many miRNAs. Identification of these miRNA‐target pairs is crucial to understanding the biology of the miRNA regulatory mechanism.
In plants, miRNAs exert negative regulatory control by binding to and causing cleavage of their targets at the position between nucleotides 10 and 11 of the miRNA. Recently, degradome sequencing, a modified 5′ rapid amplification of cDNA ends (RACE) performed with next‐gen sequencing, has emerged as a comprehensive method of analyzing patterns of RNA degradation. Deep sequencing of the 5’ ends of RNA degradation products allows identification of over‐ represented 5’ ends (miRNA cleavage sites) within an RNA sample Matching cleavage sites to known miRNA sequences links miRNAs to their targets.
Therapeutic Potential of microRNAs in Toxicogenomics
MicroRNAs (miRNAs) are effectors of environmental influences on gene expression. Thus miRNAs play an important role in the cellular response to injury, exposure to toxicants, and disease. Insight into the mechanisms through which these small molecules function may lead to development of therapeutics that target miRNA pathways. Identification and quantitation of miRNA expression is an important first step in understanding the potential mechanisms involved in these cellular and molecular responses. Here we present an advanced microfluidic biochip technology that was developed to enable comprehensive miRNA expression profiling. Detection of miRNA using an array offers the opportunity to examine all known and/or predicted miRNA transcripts in a single experiment providing a comprehensive view of all miRNAs that may be involved in the system being investigated. The detection probes are in situ synthesized using PGR (Photo-Generated Reagent) chemistry to afford the high synthesis yield and complete sequence flexibility, and modified nucleotides are incorporated to enhance the binding to short miRNAs without sacrificing specificity. Several key parameters of the biochip technology, including detectivity, specificity, and feature uniformity will be demonstrated. Application examples will be provided to demonstrate how the technology is enabling breakthrough discoveries.
miRFocus for Integration of miRNAome and Proteome Analysis
MiRNAs have now been accepted as effective molecular signatures of disease diagnosis, specifically cancers, which still remain a major threat for human vitality. In recent development of clinical technologies, our interests reside in pathway-based integrated analysis of cellular proteins and miRNAs. We developed miRFocus, a web resource, which promises to provide cancer biomarker miRNAs for cancer screening Abundant information exists linking screening. signature miRNAs with a particular cancer or a subtype of a cancer. In this presentation, we will describe our method of utilizing pathway analysis for identification of specific miRNAs as reporters of tumor genesis pathways; we experimentally demonstrate the miRFocus findings. The method has great potential in defining a molecular diagnostic (MDx or miRDx) panel for populationbased preventive examination of cancer occurrences. miRNAs are defined as regulators of target proteins. The aforementioned pathway biomarker miRNAs lead to protein candidate markers that are detected on our Proteomic PepArrays. We carried out whole cancer cell lysate analysis on the pepArrays specifically designed to assay the pathway reporter proteins. The integrated analysis of miRNA and proteomic protein profiling using bioinformatics and genomic and proteomics technologies. Differential expressed. miRFocus URL: http://mirfocus.org/
Precise Control of Diversity for Synthetic Antibody Library Design & Construction
Synthetic antibody libraries have proven to be effective tools for drug discovery and development through the generation of functional, high-affinity antibodies against a wide variety of antigens. They are an expanding alternative to standard hybridoma technology especially for application to particularly difficult therapeutic challenges that cannot be addressed with antibodies from the natural repertoire. The performance of a synthetic antibody library depends in large part on the diversity of the library which must be designed based on thorough understanding of the antibody structure and function. Focused diversity can provide an efficient path to antibody candidates designed for exceptional performance in specialized applications if precise control over design and construction is exercised. The use of degenerate oligos and other standard methods of diversity introduction lack this control and can introduce unwanted or useless codons into the library, thus limiting its performance. Fully designed library diversity is enabled through parallel in-situ (on-chip) in on synthesis of tens of thousands of specific (non-degenerate) oligonucleotide sequences We demonstrate the bioinformatics-based design and high-throughput synthesis of a mutant sequences. across the chip phage display library to improve affinity of anti-ErbB2 single chain monoclonal antibody A21.
Micro/Small RNA Detection on a Microfluidics Microchip
Detection of micro and small RNAs using a microarray offers the opportunity to examine all known and/or predicted micro/small RNA transcripts in a single experiment. A successful micro/small RNA microarray detection system consists of a high quality microarray platform, a reliable sample preparation and labeling process, and a comprehensive data analysis capability. Additionally, micro/small RNA detection is a rapidly expanding field. A detection system must be highly flexible to be able to serve the changing needs. Here we present an advanced μParaflo™ microfluidics microchip technology that was developed to enable a comprehensive micro/small RNA microarray service. Modified nucleotides are incorporated into the detection probes to enhance their binding to short micro/ small RNAs without sacrificing specificity. The detection probes are in situ synthesized using PGR (Photo-Generated Reagent) chemistry to afford the highest synthesis yield and complete flexibility in the sequences synthesized. This capability has proven to be highly valuable to scientists in their discovery studies of new micro/small RNAs and their biogenesis and functional mechanisms. The high quality of the μParaflo™ array microchips will be demonstrated by several key parameters, including detectivity, specificity, and feature uniformity. Diverse application examples will be provided to illustrate the usefulness of this highly sensitive, specific, flexible, high dynamic range, and low noise microarray technology in the evolving fields of micro/small RNA research.