Towards Providing Personalized Medicine—Considerations for Reliable Data

Scientists at Geneseeq Technology, Inc. demonstrate how they improved their target capture methods to increase accuracy in clinical diagnostics by using optimized blocking oligos and stringent hybridization conditions.

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Discriminating Highly Similar Transcripts Using rhPCR

Description of RNase H-dependent PCR, a technology developed to increase PCR specificity and eliminate unwanted interactions between primer sets (e.g., primer-dimers, etc.). An example is provided in which it is used to distinguishing highly similar alternatively spliced sequences. This technology can also be useful in genotyping applications, in highly multiplexed qPCR assays, library construction for Next Generation DNA Sequencing, and for rare allele detection, where the added specificity provided by the blocked-cleavable primers enables detection of a rare mutant allele in a background of large amounts of wild type DNA.

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Using DsiRNA to Map Pain Pathways in the CNS

DsiRNAs (Dicer-substrate RNAs) are chemically synthesized 27mer duplex RNAs that have increased potency in RNA interference compared to traditional 21mer siRNAs. Read on for an example of a lab using this technology for in vivo delivery to the CNS.

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Interpreting Melt Curves: An Indicator, Not a Diagnosis

Examining PCR melt curve data to determine what it can/cannot tell us about resulting PCR amplicons.

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Your Probe and Primer Sequences Are Always Provided

IDT always provides primer and probe sequences upon order placement, as per the MIQE Guidelines.

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RxnReady Oligos—Get Your Oligos Premixed!

Have 2–6 standard desalted DNA oligonucleotides premixed in a single tube according to your specifications. This can be useful when performing multiplex PCR, or when generating sets of insertions or deletions through site-directed mutagenesis.

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Recommended Dye Combinations for Multiplex qPCR

Recommendations for selecting dyes for multiplex qPCR that minimize background and avoid overlap of fluorescent signals. Included is a table of compatible dyes for multiplexing on common qPCR instruments and a list of suggested quenchers.

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Epigenetic Biomarkers for Prostate Cancer

Methylation and expression analysis methods are used to evaluate epigenetic markers for early, noninvasive detection of aggressive prostate cancer. IDT PrimeTime® qPCR Assays, ZEN™ Double-Quenched Probes, and gBlocks® Gene Fragments are used to facilitate this research.

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My Oligos Have Arrived: Now What?

Recommendations for resuspension and storage of newly received oligonucleotides.

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Optimized Immune Profiling Using a Synthetic Immune System

A high-throughput method for sequencing and quantification of rearranged antigen receptors on T- and B-cells using gBlocks Gene Fragments to create a “gold standard synthetic immune system” where the binding sites for every possible forward and reverse primer combination for a given receptor type were represented.

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Insertion Site Detection and Targeted RNA Capture Using Next Generation Sequencing

Scientists at Cofactor Genomics use in-solution hybridization to focus on of regions of interest for next generation sequencing

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Digital PCR (dPCR)—What Is It and Why Use It?

General overview of dPCR and how it can be used for qPCR applications, including multiplex qPCR.

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Understanding Melting Temperature (Tm)

Advice from our own Dr Richard Owczarzy on considerations for better oligo design: oligo concentration, salt, SNPs.

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Designing PCR Primers and Probes

General guidelines for designing primers and probes and for choosing target locations for PCR amplification.

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Calculations: Converting from Nanograms to Copy Number

Calculation often used when creating a qPCR standard curve. Link to free, online tool that will do it for you.

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3D DNA Canvas—Synthetic DNA Learns New Tricks

Scientists assemble DNA bricks (32mers) to create complex, nanostructure shapes, including 3D structures.

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Beginning Genotyping Experiments?

Small size of LNA Probes—enough for 100 rxns—ideal for preliminary experiments. Less cost and waste.

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qPCR Probes—Get the Right Scale at the Right Price

FAM-labeled, double-quenched qPCR probes at a mid-range scale for researchers who only need 100–500 amplification reactions – cheaper than larger reaction size.

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Exon Numbering—Not As Easy As 1, 2, 3...

Exon numbering and location data can differ across various software tools, including with NCBI's gene database. For example, exons within alternatively spliced transcripts are sometimes individually numbered, with no consistent gene-based numbering system across these transcripts for identifying exons. Here we describe how IDT exon location information gives each exon a unique number and how that compares with the NCBI naming system.

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Two Quenchers Are Better Than One!

Data demonstrating that ZEN™ Double-Quenched Probes in qPCR assays provide increased signal detection and greater assay sensitivity than single-quenched probes, such as probes with BHQ Quenchers. ZEN™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.

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Innovative Therapeutic Antibodies

Use of gBlocks® Gene Fragments to synthesize antibody variable regions to create novel heavy/light chain combinations.

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GAPDH, a Good Reference Sequence?

Housekeeping genes commonly used as internal controls, often may not be the best choices due to lesser known functions and the presence of pseudogenes, some of which, in the case of GAPDH---used here as an example--may be expressed.

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Easily-Designed Standard Curves for qPCR

An easy way to combine control templates/multiple targets onto a single construct and the advantages that can provide for PCR experiments.

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Oligopaints—Visualizing the Three-Dimensional Organization of the Genome

Use of modified oligos as probes for chromosomal mapping of specific sequence elements and how their location correlates with the 3D organization of the genome.

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When Designing PCR Assays, Always Check For SNPs

With the increasing identification of SNPs, it is critical to check whether they underlie your primer or probe sequences, which could compromise PCR efficiency.

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Digital PCR—Simplifying Quantitative PCR

Theory behind why digital PCR makes gene expression quantitation easier & more accurate, and how Raindance’s use of ZEN & LNA probes has increased their signal-to-noise ratio and rare allele detection.

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Synthetic Life: Dr Dan Gibson Discusses the Science of Big DNA

Q&A style interview with Dr Dan Gibson discussing approaches for assembling synthetic, genome-sized DNA.

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DNA Methylation Analysis—Keeping it Simple

Use of methylation-sensitive restriction enzymes and probe-based PCR to provide % methylation for specific amplicon regions (primarily promoters). Read how IDT double-quenched probes, PrimeTime qPCR Assays, and gBlocks Gene Fragments augment this analysis.

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Target Enrichment Identifies Mutations that Confer Fitness Effects

Target enrichment using xGen® Lockdown® Probes and next generation sequencing were used to track the frequency of mutations in evolving bacterial populations over a given time course and to gauge their importance based on their fitness effect.

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PrimerQuest® Design Tool: From Basic to Highly Customizable Designs

When and how to use the PrimerQuest Design Tool to customize primers and probe for a wide variety of qPCR applications. Examples show how to adjust reaction conditions, add a probe to a set of previously designed primers, define primer positions, and include or exclude sequences from the assay designs.

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Multiplex qPCR—How to Get Started

Learn how multiplex qPCR can save sample, reagent cost, and time. The article provides recommendations for multiplex qPCR assay design and experimental setup.

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Improving Vaccine Development

A directed, molecular evolution process employed by the biopharmaceutical company, Altravax, uses in vitro DNA recombination to generate large libraries of recombined, chimeric DNA sequences that express potential vaccine candidates. IDT gBlocks Gene Fragments have proved instrumental in this high throughput technology.

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Undergraduate iGEM Competition Moves Synthetic Biology Forward

An overview of the 2012 top 4 highest placing teams in the International Genetically Engineered Machine competition (iGEM) with a more in depth summary of 2nd place winner Slovenia's project on an engineered inducible cellular switch (Switch-it system) for delivery of therapeutic drugs directly to target tissues.

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Small RNA Therapies for Cystic Fibrosis

The McCray Lab (Univ IA, USA) used DsiRNAs to develop a protocol for efficient small RNA delivery into airway epithelia. The resulting method was then used to study miRNA regulation of gene products involved in airway fluid and electrolyte transport, cellular mechanisms linked to cystic fibrosis.

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Building Biological Factories for Renewable and Sustainable Products

Amyris uses genetic engineering and screening technologies to design microorganisms that convert plant-sourced sugars into target molecules, including pharmaceuticals, biofuels, polymers, flavors, and fragrances.

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Generating Quick Constructs for Intracellular Vesicular Transport Studies

gBlocks Gene Fragments provide rapid and cheap access to new types of functional and structural elements for constructing novel biological modules and cascades to better understand the tethering complexes and transcription-regulating complexes critical to intracellular vesicular transport.

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Next Generation Sequencing in the Clinic: A Perspective from Dr Elaine Mardis

Dr Elaine Mardis shares her views on the current uses of NGS, the challenges that NGS technologies face, and what can be expected in the future.

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Easy Resuspension and Dilution of Oligonucleotides

Screen shots of updated calculation tools and note of the variety of units that can be used as input values.

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Single-Nucleotide Resolution of RNA Structure

Shape chemical reagents differentially modify accessible nucleotides in an RNA molecule, and then be mapped to elucidate the 3D structure of even very large RNAs.

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A Next Generation Understanding of Immune Response

Assembly PCR of antibodies with gBlocks™ Gene Fragments and PCR-amplified DNA to study the immune system's response to patients receiving flu vaccinations.

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Fast and Accurate Assembly of a 2.1 kb Gene

Fast, cost-effective assembly of genes using gBlocks Gene Fragments and Gibson Assembly Master Mix.

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Genomic Target Selection Using Individually Synthesized Capture Probes

Biotinylated Ultramer™ Oligonucleotides perform better than array-synthesized probes during target capture for next generation sequencing.

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qPCR Terminology—What Does It Mean?

Definitions of some of the most commonly used terms and distinctions encountered in qPCR experiments.

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Barcoding Life

The International Barcode of Life Project—construction of a comprehensive database of DNA sequence tags for eukaryotic life that links genetic, morphological, and ecological data.

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iGEM—Extending the Reach of Synthetic Biology

Four teams talk about their creative applications of synthetic biology methods for the International Genetically Engineered Machine (iGem) competition.

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Towards Real-Time Imaging in Single, Living Cells

Developing oligonucleotide technologies for detection and quantification of specific RNA transcripts in live cells.

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qPCR with Intercalating Dyes: PrimeTime® qPCR Primers

PrimeTime qPCR Primers are now available to detect genes in human, mouse, and rat transcriptomes with intercalating dyes such as SYBR® Green and EvaGreen®

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Calculation Tips for Resuspending and Diluting Nucleic Acids

Easy guidelines for making a 100 uM solution; calculating nmoles, ug, copy number, and concentration; and determining concentration equivalencies

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A Conversation About qPCR with Jo Vandesompele

An interview with Dr Jo Vandesompele, internationally recognized expert on quantitative PCR, discussing the common issues researchers face when using qPCR, and the future direction of qPCR technology.

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Sample Preparation for Successful qPCR

Considerations for qPCR sample preparation for obtaining accurate and consistent results.

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The Effect of Genetic Variation on Phenotype

Development of a Targeted Genome Enrichment (TGE) technique to simultaneously sequence genomic regions of patients exhibiting the same phenotype.

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Steps for a Successful qPCR Experiment

This article will focus on 5′ nuclease assay design and experimental setup considerations that will assist in obtaining accurate and consistent results.

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Assay Design Tools for qPCR

IDT offers a group of qPCR design tools to suit your specific application needs. Whether you want to quickly order a predesigned assay, or desire assistance with designing assays for a challenging target, IDT has a design tool that can help.

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Starting with RNA—One‑Step or Two‑Step RT‑qPCR?

When performing real-time qPCR, one has to decide whether to use a one-step protocol that combines the RT reaction and PCR in one tube, or a two-step protocol where the RT reaction is performed separately from the PCR.

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High Throughput Sequencing of Genomic Interval Involved in Fetal Growth Restriction

Use of Targeted Solution Hybrid Capture, Hamming Barcoding, and NGS

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Identifying Stem Cell Differentiation Biomarkers

MultiStem have greater differentiation potential than typical mesenchymal stem cells.

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Using Site-directed Mutagenesis to Elucidate Structure: Function Relationships

Small Noncoding RNAs and Stress-induced Transcription Factors

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The Future of RNAi and Aptamer Technologies

An interview with Dr John Rossi, Chair and Professor of the Department of Molecular and Cellular Biology at the Beckman Research Institute of City of Hope (Duarte, CA). His research focuses on the biology and applications of eukaryotic small RNAs and, in particular, their therapeutic use in HIV/AIDS and cancer.

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Choosing a qPCR Assay: Inventoried or Predesigned?

Several companies offer inventoried qPCR assays that have not only been predesigned, but are also premanufactured and stocked, waiting for that order to be placed. In contrast, IDT PrimeTime Pre-designed qPCR Assays are not pre-manufactured and have undergone rigorous bioinformatics assessments using sophisticated design algorithms to generate the most efficient assays for a particular genetic region.

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Using PrimeTime qPCR Assays in Multiplex Experiments

IDT PrimeTime qPCR Assays offer multiple dye/quencher combinations and primer/probe ratios to simplify the multiplex experiment design.

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Tips for Using BLAST to Locate PCR Primers

Need a quick way to find the location of primers within a gene or the expected size of the resultant PCR product? In this tip we show you how to get this information using BLAST.

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Screening SNPs with Millions of Oligos

Curtis Kautzer is the consummate IDT customer, having used IDT products in numerous applications through his career (primers for long range sequencing of the human genome, extra long oligos as PCR assay targets, adapters for DNA sequencing).

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Demystifying Ribosome Assembly with Adenylated Oligos

Ribosomes assemble proteins—fundamental components of cells. A typical yeast cell contains over 200,000 ribosomes and makes about 2000 every minute.

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Exploring and Exploiting Ion Channel Function

Using custom synthesized MiniGene constructs to make peptides.

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