Easily-Designed Standard Curves for qPCR

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 of 125–750 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 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:

  1. 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.
  2. 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.

For more information about gBlocks Gene Fragments, or to order, go to www.idtdna.com/gBlocks.


Related Reading

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. Read about it in DECODED 2.1 at www.idtdna.com/decoded.

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. Read about it in DECODED 3.2 at www.idtdna.com/decoded.

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. Read about it in DECODED 3.1 at www.idtdna.com/decoded.

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. Read about it in DECODED 2.4 at www.idtdna.com/decoded.

Undergraduate iGEM Competition Moves Synthetic Biology Forward

Description of Slovenia’s 2012 iGEM Team project on an engineered inducible cellular switch (Switch-it System) for delivery of therapeutic drugs directly to target tissues. Read about it in DECODED 3.2 at www.idtdna.com/decoded.

 

Author: Hans Packer, PhD, is a Scientific Writer at IDT.