Direct PCR from human nail DNA sample

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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 Conclusions Methods

Introduction

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.

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).

Comparison of expression levels based on GC-content

Amplification results for Terra Direct compared to two other DNA polymerases
Lane No.	Target gene	Amplicon size	% GC content	Amplification results
Lane No.	Target gene	Amplicon size	% GC content	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	40 cycles

Related Products

Cat. #	Product	Size
639270	Terra™ PCR Direct Polymerase Mix	200 Rxns	USD $172.00
A hot-start polymerase mix that is developed for direct PCR amplification from tissue samples, crude extracts, and dirty templates. It is ideal for amplifying short DNA targets (up to 2 kb), regardless of GC content or template purity. The polymerase mix is supplied with separate tubes of buffer (with Mg²⁺ and dNTPs) and Proteinase K. The inclusion of Proteinase K enhances gel detection of the PCR products. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components Image Data Resources Back Terra PCR Direct was used to amplify the cyclin D2 gene (Ccnd2, 0.5 kb; Lane 1) and the transferrin receptor gene (TfrC 2 kb; Lane 2) from 1 μl of mouse blood treated with either EDTA or heparin Panel A. Terra PCR Direct was used to amplify the cyclin D2 gene (Ccnd2, 0.5 kb; Lane 1) and the transferrin receptor gene (TfrC, 2 kb; Lane 2) from 1 μl of mouse blood treated with either EDTA or heparin. Panel B. Terra PCR Direct was used to amplify the mouse Ywhaz1 gene (1 kb) directly from either a 1 mm tail or 1.5 mm² ear biopsy. A 4 μl aliquot of each sample was mixed with gel loading buffer that either lacked or contained proteinase K (Lanes 1 and 2, respectively). The PCR products treated with proteinase K ran as expected, whereas those without proteinase K treatment got stuck in the wells. Panel C. Terra PCR Direct was used to amplify the cytochrome c oxidase gene (cox1; 0.5 kb) directly from 0.5 mm (Lane 1) and 1.2 mm (Lane 2) tomato or spinach leaf cuttings (made using hole punches). Back 639270: Terra PCR Direct Polymerase Mix
639271	Terra™ PCR Direct Polymerase Mix	800 Rxns	USD $573.00
A hot-start polymerase mix that is developed for direct PCR amplification from tissue samples, crude extracts, and dirty templates. It is ideal for amplifying short DNA targets (up to 2 kb), regardless of GC content or template purity. The polymerase mix is supplied with separate tubes of buffer (with Mg²⁺ and dNTPs) and Proteinase K. The inclusion of Proteinase K enhances gel detection of the PCR products. Notice to purchaser Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Documents Components Image Data Resources Back Terra PCR Direct was used to amplify the cyclin D2 gene (Ccnd2, 0.5 kb; Lane 1) and the transferrin receptor gene (TfrC 2 kb; Lane 2) from 1 μl of mouse blood treated with either EDTA or heparin Panel A. Terra PCR Direct was used to amplify the cyclin D2 gene (Ccnd2, 0.5 kb; Lane 1) and the transferrin receptor gene (TfrC, 2 kb; Lane 2) from 1 μl of mouse blood treated with either EDTA or heparin. Panel B. Terra PCR Direct was used to amplify the mouse Ywhaz1 gene (1 kb) directly from either a 1 mm tail or 1.5 mm² ear biopsy. A 4 μl aliquot of each sample was mixed with gel loading buffer that either lacked or contained proteinase K (Lanes 1 and 2, respectively). The PCR products treated with proteinase K ran as expected, whereas those without proteinase K treatment got stuck in the wells. Panel C. Terra PCR Direct was used to amplify the cytochrome c oxidase gene (cox1; 0.5 kb) directly from 0.5 mm (Lane 1) and 1.2 mm (Lane 2) tomato or spinach leaf cuttings (made using hole punches).

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