Terra PCR Direct Polymerase Mix contains an optimized DNA polymerase that allows for direct amplification from a tissue source or crude extracts, without the need for DNA purification. It can amplify DNA targets up to 4 kb as well as difficult templates (including those with a GC content greater than 70%)—and it enables DNA amplification in the presence of PCR inhibitors, growth serum, and other source-material components.
Tech Note
Amplifying a gene of interest from human nail DNA
Terra PCR Direct outperforms competitor PCR enzymes for direct amplification of GC-rich targets and longer (4 kb) targets
- Overview: Terra PCR Direct Polymerase Mix contains an optimized DNA polymerase
- Comparison with competitors’ PCR enzymes: Terra PCR Direct Polymerase demonstrates an exceptional ability to amplify GC-rich targets
Introduction
Results
Comparison with competitor PCR enzymes
Terra PCR Direct Polymerase Mix demonstrates an exceptional ability to amplify GC-rich targets and relatively long (4 kb) targets from a crude extract containing human nail DNA.
Terra PCR Direct Polymerase Mix (Panel A) was able to successfully amplify all of the samples. The PCR enzyme from Company A (Panel B) was unable to amplify any of the samples, except for slight amplification of one AT-rich sample (Panel B, Lane 4). The PCR enzyme from Company B (Panel C) was unable to amplify any of the GC-rich samples (Panel C, Lanes 3, 6, and 9) and exhibited diminished performance in amplifying targets as target length increased (Panel C, Lanes 2, 5, and 8).
Comparing the ability of three DNA polymerases to amplify target gene fragments of various sizes and % GC content from a crude human nail DNA extract using agarose gel electrophoresis. Terra PCR Direct polymerase was able to amplify gene fragments from GC-rich templates up to 4 kb in length (Panel A), while the Company A enzyme provided poor or no amplification, except for one AT-rich sample (Panel B), and the Company B enzyme failed to amplify gene fragments containing over 50% GC (Panel C). For each image, Lane M contains the pHY Marker.
| Amplification results for Terra Direct compared to two other DNA polymerases | ||||||
|---|---|---|---|---|---|---|
| Lane No. | Target gene | Amplicon size | % GC content | Amplification results | ||
| Terra PCR Direct | Company A enzyme | Company B enzyme | ||||
| 1 | UCRchr9* | 1.1 kb | 26% | + | – | + |
| 2 | EGFR | 1.0 kb | 50% | + | – | + |
| 3 | TGFβ1 | 1.0 kb | 72% | + | – | – |
| 4 | UCRchr11** | 2.2 kb | 27% | + | + | + |
| 5 | IGF2R | 2.0 kb | 50% | + | – | + |
| 6 | IGFβ1 | 2.0 kb | 69% | + | – | – |
| 7 | FragileX | 4.0 kb | 33% | + | – | + |
| 8 | EGFR | 3.8 kb | 44% | + | – | – |
| 9 | TGFß1 | 4.0 kb | 63% | + | – | – |
*UCRchr9 = Chromosome 9 AT-rich noncoding region.
**UCRchr11 = Chromosome 11 AT-rich noncoding region
Conclusions
This experiment demonstrates that Terra PCR Direct Polymerase is a good choice for analyzing extremely tough, hard-to-lyse tissues such as human nails, which are frequently the subject of forensic analysis. Terra was able to amplify gene fragments from GC-rich templates up to 4 kb in length. In contrast, the polymerase from Company B failed to amplify gene fragments containing over 50% GC, while the polymerase from Company A provided poor or no amplification, except for one AT-rich sample.
Methods
A crude extract containing human nail DNA was prepared by adding 44 μl of SimplePrep reagent mix (not sold in the U.S.) to a 5-mg human nail sample and treating it according to the standard SimplePrep protocol. 2.5-μl aliquots of the human nail DNA extract were then used as the template in a 25-μl reaction volume to amplify different target gene fragments ranging from 1–4 kb in length and with 26–72% GC content, using Terra PCR Direct Polymerase Mix or inhibitor-resistant PCR enzymes from Companies A and B as shown in the table above. Amplification reactions were performed with each enzyme according to the manufacturer’s instructions, and 3-μl samples of each amplification reaction were analyzed on an agarose gel as shown in the figure above.
The PCR reactions using Terra PCR Direct Polymerase Mix were performed according to the following program:
| 98°C | 2 min | |||
| ↓ | ||||
| 98°C | 10 sec | 40 cycles | ||
| 68°C | 1 min/kb |
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