gBlocks® Gene Fragments—Related DECODED Articles

Predictable control of gene expression by mRNA, 3’ untranslated region motifs

Yoon OK, Hsu TY, et al. (2012) Genetics and regulatory impact of alternative polyadenylation in human B-lymphoblastoid cells. PLoS Genet, 8(8):e1002882; doi:  10.1371/journal.pgen.1002882.

Yoon et al. examined the regulatory impact of mRNA 3’ untranslated region motifs. The authors performed qPCR experiments using gBlocks® Gene Fragments to generate DNA standard curves for absolute quantification of mRNA in cells transfected with 3’ UTR reporters. This work demonstrates that double-stranded gBlocks Gene Fragments can serve as an ideal source for qPCR standards. They can be ordered up to 3000 bp in length, are normalized to 200–1000 ng, depending on size, and are sequence-verified. Several amplicons can be designed into a single gBlocks Gene Fragment sequence for multiplex amplifications. As an added benefit, because they are limited in quantity and do not require additional processing and purification, gBlocks Gene Fragments can reduce the risk of template contamination in the lab that is common with plasmid DNA.

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PrimeTime® qPCR Assays

  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies

  • Primer-based assays—designed for intercalating dye experiments

Create custom assays that are designed using our proprietary bioinformatics algorithms for any target and to your specific parameters. Alternatively, select one of our predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence/SNP information.

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Double-Quenched Probes

ZEN and TAO Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and Iowa Black® FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.

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gBlocks® Gene Fragments

gBlocks Gene Fragments are double-stranded, 125–3000 bp DNA molecules. They are ideal for use as qPCR controls and standards, as well as for gene construction and editing applications. These affordable gene fragments are sequence-verified, ship in a few working days, and save laboratory time.

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Additional reading

Easily-designed standard curves for qPCR—Learn how to use synthetic gBlocks Gene Fragments for creating standard curves, including incorporating multiple targets into a single gene fragment.

Multiplex qPCR—how to get started—Review tips for setting up multiplex qPCR experiments.

Increase sensitivity and precision in your qPCR experiments—Use double-quenched probes to decrease background, and increase sensitivity and precision in your qPCR experiments.

Recommended dye combinations for multiplex qPCR—Read these recommendations for selecting dyes for multiplex qPCR to 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.

Creating a synthetic immune system for optimized immune profiling—Find out how researchers at Adaptive Biotechnologies used gBlocks Gene Fragments to optimize a complex, multiplex PCR for sequencing and quantification of rearranged antigen receptors on T- and B-cells.

Optimizing multiplex qPCR for detecting infectious diseases and biothreat agents in the field—Researchers at Tetracore specialize in developing large sets of robust probe-based qPCR assays for use in a multiplex format to detect infectious diseases and bio-terrorism threat agents. Here they discuss the need to: use probe dyes compatible on common PCR instruments, maintain low background with multiple probes, and reformulate assays to address viral mutation; and how ZEN Double-Quenched Probes have helped meet these criteria.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Hans Packer, PhD, is a scientific writer at IDT.

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