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, a new alternative to single-stranded oligonucleotides, enables 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 citations box on the right.
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.