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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 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 of greater than 70%)—and it enables DNA amplification in the presence of PCR inhibitors, growth serum, and other source material components.

Comparison with Competitor PCR Enzymes

Terra PCR Direct Polymerase 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 (Lane 4). The PCR enzyme from Company B (Panel C) was unable to amplify any of the GC-rich samples (Lanes 3, 6, and 9) and showed increasing difficulty in amplifying targets as the length of the target increased (Lanes 2, 5, and 8).

Comparison of expression levels based on GC-content

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). Lanes M denote 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 TGFß1 1.0 kb 72% +
4 UCRchr11** 2.2 kb 27% + + +
5 IGF2R 2.0 kb 50% + +
6 IGFß1 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

Summary

This experiment demonstrates that Terra PCR Direct Polymerase is a good choice for analyzing extremely tough, hard-to-lyse tissues such as human nail, 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. Then 2.5 μl aliquots of the human nail DNA extract were used as the template in a 25 μl reaction volume to amplify different target gene fragments ranging from 1–4 kb in length and 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:

Terra Human Nail diagram

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