Creating artificial templates using gBlocks® Gene Fragments
Templates for absolute quantification
When performing quantitative PCR (qPCR), absolute quantification is usually accomplished by including artificial templates such as plasmids, oligonucleotides, or purified PCR products that have been accurately quantified by independent analysis. Standard curves plotted to known concentrations are then created by performing qPCR on serial dilutions of these templates.
Plasmids that have been sequenced are excellent for generating standard curves, but can be costly and time consuming to produce. Oligonucleotides and PCR products can be produced quickly, allowing greater flexibility when changing assays, but are limited in size. Additionally, PCR products may have unidentified sequence errors that will alter the efficiency calculation that is necessary during absolute quantification.
Double-stranded gBlocks Gene Fragments
Double-stranded, sequence-verified gBlocks Gene Fragments are a new alternative to single-stranded oligonucleotides that enable creation of long, completely custom DNA sequences. Available in lengths up to 3000 bp, they function exactly like a double-stranded PCR product in applications, while offering all of the sequence flexibility of custom, chemically synthesized DNA. Furthermore, by including necessary sequence overlaps or restriction sites, gBlocks Gene Fragments are ideally suited for isothermal assembly or cloning into any plasmid, providing a mechanism for production of additional high-fidelity template. To read about these and other gBlocks Gene Fragment applications, see the Related reading box below.
Generating standard curves from gBlocks Gene Fragments—selected citations
Greiman SE, Tkach VV. (2016) The numbers game: quantitative analysis of Neorickettsia sp. propagation through complex life cycle of its digenean host using realtime qPCR. Parasitol Res, 115(7):2779–2788.
Lee J, Foong YH, et al. (2016) Analysis of specific RNA in cultured cells through quantitative integration of q-PCR and N-SIM single cell FISH images: application to hormonal stimulation of StAR transcription. Mol Cell Endocrinol, 429:93–105.
Perry AS, Baird AM, Gray SG. (2015) Epigenetic Methodologies for the study of Celiac Disease. Methods Mol Biol, 1326:131–158.
Sandkam B, Young CM, Breden F. (2014) Beauty in the eyes of the beholders: Color vision is tuned to mate preference in the Trinidadian guppy (Poecilia reticulata). Mol Ecol, 24(3):596–609.
Gunawardana M, Chang S, et al. (2014) Isolation of PCR quality microbial community DNA from heavily contaminated environments. J Microbiol Methods, 102:1–7.
Generating multiple standard curves from a single template
The design flexibility of gBlocks Gene Fragments is uniquely advantageous when incorporating multiple control amplicon sequences into a single double-stranded construct. Using gBlocks fragments as multi-control templates lowers cost to a level that is comparable to ordering individual oligos on a per assay basis. It also provides some unique benefits when performing qPCR experiments in the lab:
- When performing multiplex experiments, combining control templates onto a single construct means less pipetting and, thus, less experimental variability. Each assay on that construct will have exactly the same amount of template available, providing for more accurate comparisons between those assays.
- For singleplex reactions, you need only make one set of dilutions. Those dilutions can then be used for all the assays represented on that construct. Again, this reduces the chances for pipetting error, and saves time diluting multiple, distinct templates.
When designing gBlocks Gene Fragments with multiple targets, some researchers choose to separate each sequence with several intervening T bases, as shown in Figure 1. However, do not add more than 9 T bases between sequence elements, as this will interfere with the manufacture of your gBlocks fragment.
Figure 1. Easily incorporate multiple controls into one gBlocks® Gene Fragment. In this example, 4 sequences are incorporated into 1 design, each separated by 5 intervening T bases (red). A NotI restriction site (blue) is included in this design to provide a future site for plasmid linearization.
Use gBlocks Gene Fragments to detect contamination
Another benefit of gBlocks Gene Fragments for qPCR is the ability to quickly generate artificial sequences that can be distinguished from wild-type sequences. In Figure 2, an artificial construct that is 10 bp shorter than the wild-type sequence (LIMK1(–10)) can be distinguished by performing melt curve analysis using intercalating dye–based assays, such as with SYBR® Green. This is extremely beneficial if you have any concerns about possible contamination by wild-type DNA.
Figure 2. Designing artificial control sequences can help identify contaminating DNA. In this example, which uses a SYBR® Green dye–based assay, the artificial LIMK1(–10) sequence is easily distinguished from a wild-type sequence by the lower peak on this melt curve analysis.
Product focus: qPCR reagents and custom dsDNA fragments
qPCR reagents—everything but your sample
All the reagents you need for successful qPCR assays are available through IDT.
- Master mix
- Both probe-based and primer only qPCR assays
- Water and buffer
Custom dsDNA fragments—gBlocks® Gene Fragments
These double-stranded, sequence-verified, DNA genomic blocks, 125–3000 bp in length, are designed by you, and are shipped in 2–5 working days for affordable and easy gene construction or modification. They have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.
Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.
Steps for a successful qPCR experiment—Read these recommendations for 5′ nuclease assay design and experimental setup that will help you obtain accurate and consistent results.
How to avoid false positives in PCR and what to do if you get them—Do you know what causes false positives in the Negative Template Control sample during PCR? Review the causes and get these suggestions for preventing them.
Designing PCR primers and probes—Many factors can influence successful PCR experiments, including primer and probe location, length, interaction and self-folding, melting temperature, annealing temperature, and GC content. Review these general recommendations for designing primers and probes and for choosing target locations for PCR amplification.
Isothermal assembly: Quick, easy gene construction—Learn how, in a single reaction, isothermal assembly can combine several overlapping DNA fragments to produce a ligated plasmid ready for transformation.
Building biological factories for renewable and sustainable products—Read how 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.
A next generation understanding of immune response—See how assembly PCR of antibodies with gBlocks Gene Fragments and PCR-amplified DNA is used to study the immune system’s response to patients receiving flu vaccinations.
Review other DECODED Online newsletter articles on PCR and qPCR applications, and gBlocks Gene Fragment use in synthetic biology applications.
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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