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 Competitor PCR Enzymes:
Terra PCR Direct Polymerase demonstrates an
exceptional ability to amplify GC-rich 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).
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
Amplification Results
Lane
No.
Target
Gene
Amplicon
Size
% GC
Content
1
UCRchr9*
1.1 kb
2
EGFR
1.0 kb
Terra
PCR
Direct
Company A
Enzyme
Company B
Enzyme
26%
+
–
+
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:
http://www.clontech.com/US/Support/Applications/PCR/Direct_PCR_Human_Nail_Data