Core Concepts
Scientific Fundamentals Explained

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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Modification Highlight: Modifications that Block Nuclease Degradation

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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Modification Highlight: When dT is Required For Modification Attachment

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

Useful modifications and design considerations for effective antisense oligonucleotides.

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Modification Highlight: ZEN™ Internal Quencher

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

We explain 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

What are G-quadruplexes and how do they 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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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 base pairs.

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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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Modification Highlight: Digoxigenin

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

Tips and tricks for blunt-end cloning.

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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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Modification Highlight: Photo-Cleavable Spacer

How photocleavable spacers can be used in your research.

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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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Modification Highlight: Phosphorothioates

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

RNA interference (RNAi) is a conserved pathway found in most eukaryotes where dsRNAs suppress expression of genes with complementary sequences. RNAi has become the experimental tool of choice for studying the effects of gene silencing.

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Time to go GMP?

The Clinical and Commercial Manufacturing (CCM) suite is a “facility within a facility” dedicated to the GMP manufacture of oligos for diagnostic use. The CCM suite services customers who need defined specifications or require 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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Modification Highlight: Dithiol Modifier

Thiol-modified oligonucleotides are used in attachment chemistry reactions to bind an oligo to a target. Targets are commonly gold, 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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Modification Highlight: Cholesterol-TEG

Cholesterol-TEG is available from IDT for both DNA and RNA oligos on scales from 100 nmole to 10 μmole. Because cholesterol is difficult to manufacture, it requires HPLC purification. To order, select Cholesterol-TEG from the modifications tab on the oligo order page.

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