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Core Concepts
Scientific Fundamentals Explained

Use splice junctions to your advantage in qPCR

Get recommendations for avoiding PCR amplification of genomic DNA, as well as for identifying and quantifying splice variants, in this article on designing qPCR assays that span splice junctions.

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Observing subpopulations within cloned plasmids using NGS analysis

IDT is transitioning sequence verification of our Genes products from Sanger sequencing methods to NGS. Read more to find out what the benefits are when using NGS for analysis of cloned genes.

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Planning to work with aptamers?

We are often asked whether IDT manufactures aptamers. The answer is, yes! IDT does synthesize aptamers and aptamer libraries, and there are already 100s of published research papers describing the successful use of such sequences manufactured by IDT. Learn about aptamers, SELEX, and how IDT can assist you with reagents for your aptamer applications.

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CRISPR guide RNA format affects genome editing outcomes

Learn how use of different formats for the guide RNAs associated with CRISPR-Cas9 genome editing can lead to different editing outcomes. The optimized, short RNA oligos that make up the crRNA and tracrRNA components of the Alt-R™ CRISPR-Cas9 System outperform other CRISPR guide RNA formats. Unlike DNA expression constructs, short RNA oligos also are unable to incorporate into the target genome for cleaner editing results.

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Oligo synthesis: Why IDT leads the oligo industry

Read about the phosphoramidite method of oligonucleotide synthesis that IDT uses in its manufacturing processes. We also highlight the additional measures we take to ensure our customers receive the highest quality oligos and nucleic acid products in the shortest time possible.

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Antisense Oligonucleotides (ASOs)

Modification Highlight: The first oligonucleotide-based approach for disrupting gene expression—learn about new applications for antisense oligos, including the study of lncRNA function.

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Tips for successful lncRNA knockdown: Design, delivery, and analysis of antisense and RNAi reagents

IDT research scientist Kim Lennox has been optimizing effective lncRNA knockdown with antisense and RNAi reagents. Here she provides some tips for successful lncRNA knockdown.

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A new renaissance for antisense in the era of lncRNA

Noncoding RNAs such as lncRNA, are much more prevalent in humans than protein-coding RNA. Antisense oligonucleotides (ASO), previously used for knockout experiments, are being employed to study the role of noncoding RNAs in gene regulation. ASOs provide several advantages over siRNAs (and DsiRNA) for this purpose.

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Site-directed mutagenesis—improvements to established methods

Site-directed mutagenesis techniques have relied primarily on PCR and standard cloning methods. Read about some of the common cloning methods used for mutagenesis and how double-stranded DNA fragments (gBlocks Gene Fragments) can save you both time and money.

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Need a non-standard modification?

Not finding the modification you need? IDT will consider any modification you have in mind. In addition to our standard modifications, IDT offers 89 modifications that are not listed in our online catalog. A few of the more popular ones are described along with information on how to order them. Contact us with any inquiries at noncat@idtdna.com.

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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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Target enrichment facilitates focused next generation sequencing

The rationale and benefits of enriching subsets of the genome (target enrichment by hybrid capture) prior to sequencing.

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Modifications that block nuclease degradation

Modification Highlight: Description of modifications that can be added to an oligo to limit nuclease degradation, for example, when experiments where the oligos are to be used in cell culture or in vivo.

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CRISPR and Cas9 for Flexible Genome Editing

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) are sequences that serve as an adaptive defense ("immune") systems in bacteria and archaea. In conjunction with CRISPR associated (Cas) proteins, CRISPR sequences are able to recognize and cleave complementary DNA sequences. This natural mechanism has been coopted by scientists for targeted gene editing or removal. This article describes some of the early applications for which this technology is being used.

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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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When dT is required for modification attachment

Modification Highlight: Certain modifications require a dT base in the oligonucleotide sequence in order to be added.

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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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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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How to Avoid False Positives in PCR and What to Do If You Get Them

Causes of false positives in the Negative Template Control sample during PCR, and suggestions for preventing them.

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Using antisense technologies to modulate noncoding RNA function

Review useful modifications and design considerations for effective antisense oligonucleotides.

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ZEN™ Internal Quencher

Modification Highlight: Use of the ZEN Quencher as a second, internal quencher in qPCR 5’-nuclease assay probes provides greater overall dye quenching, lowering background, and increasing signal detection. When incorporated into oligonucleotides, it also serves to strengthen duplex formation and block exonuclease digestion, while remaining nontoxic to cells. Thus the ZEN Quencher can be useful in steric blocking antisense oligonucleotide applications.

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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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Oligos for molecular diagnostics

Understand the difference between Good Manufacturing Practices (GMP) and ISO 13485 certification and what these credentials mean for oligonucleotides manufactured for human diagnostics.

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G repeats—structural challenges for oligo design

Find out what G-quadruplexes are and how they can affect oligonucleotide synthesis and applications.

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DNA Oligonucleotide Resuspension and Storage

Upon receiving newly synthesized oligonucleotides, researchers must decide how to resuspend and store the product. Here are some guidelines and recommendations.

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Cloning Strategies Part 1: Assembly PCR for Novel Gene Synthesis

Single-stranded oligos or a mix of single- and double-stranded DNA are used to produce longer genes of up to several thousand bp.

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Oligo Modification—Post-Synthesis Conjugation Explained

Addition of NHS esters, and of Amino and Alkene mods through Click Chemistry. Common questions regarding post-synthesis conjugation are also answered.

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Which Biotin Modification to Use?

Applications of each of the different biotins available from IDT.

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Digoxigenin

Modification Highlight: Learn about Digoxigenin—a modification suitable for use as an alternative to biotin/streptavidin and in non-radioactive hybridization applications.

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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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Cloning Strategies Part 3: Blunt-End Cloning

Blunt-end cloning is one of the easiest and most versatile methods for cloning dsDNA into plasmid vectors. It is easy because the blunt-ended insert requires little to no preparation. Read an overview of blunt-end cloning with tips for making this cloning approach successful.

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Photo-cleavable spacer

Modification Highlight: This modification can be cleaved by a specific wavelength of UV light, fragmenting an oligo or releasing a terminal modification, such as a fluorophore.

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Oligonucleotide modifications: Choosing the right mod for your needs

Guidelines on selecting oligonucleotide modifications and how they can help you in your research.

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The Importance of Tm in Molecular Biology Applications

Predicting and selecting appropriate Tms for oligo hybridization steps (e.g. for PCR).

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Cloning Strategies Part 2: Cohesive-End Cloning

Tips and tricks for cloning using restriction enzymes.

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Isothermal Assembly: Quick, Easy Gene Construction

In a single reaction, isothermal assembly combines several overlapping DNA fragments to produce a ligated plasmid ready for transformation.

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Methods for Site-Directed Mutagenesis

Overview of the main techniques used for mutating specific gene construct regions.

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Phosphorothioates

Modification Highlight: Phosphorothioate bonds substitute a sulfur atom for one of the non-bridging oxygen atoms in the phosphate backbone of an oligonucleotide.

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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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RNAi and DsiRNA: Pathway, Mechanism, and Design

An overview of the RNA interference (RNAi) pathway, its applications, and design considerations for gene silencing experiments.

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Time to go GMP?

Learn how GMP manufacture of oligos for diagnostic use provides customers defined specifications and control over manufacturing processes.

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How biotin became a tool of molecular biologists

Biotechnology has an inclination towards co-opting nature’s most useful tools. The naturally occurring, extraordinarily strong bond that forms between avidins and biotin has provided a useful tool that has enabled numerous techniques that would otherwise be impossible.

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Understanding Mass Spectrometry of Oligonucleotides

Oligonucleotide synthesis is a complex process that requires more than one hundred sequential chemical reactions to make a single, 25-base sequence. Contemporary synthesis chemistry is robust and modern synthesis platforms are reliable and highly automated. Still, each oligonucleotide synthesized at IDT is evaluated for quality before shipping to ensure that the correct sequence was made. The best method available to assess compound identity in a high throughput environment is mass spectrometry (MS).

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Dithiol modifier

Modification Highlight: Thiol-modified oligonucleotides are used in attachment chemistry reactions to bind an oligo to a target. Targets are commonly gold molecules, but can also include a variety of fluorescent and nonfluorescent moieties.

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Unraveling RNA—the importance of a 2' hydroxyl

On paper, the small structural differences between RNA and DNA may not look substantial but, in practice, these small differences have major significance for the biological role of RNA.

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Cholesterol-TEG

Modification Highlight: Incorporate Cholesterol-TEG into your oligonucleotides to facilitate uptake into cells. This modification has been used as a transfection aid for antisense oligos and siRNAs, both in vitro and in vivo.

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