DNA from Oragene®/saliva samples† is ideal for long-range PCR
M. Keddache‡ and P. Lem**‡ DNA Sequencing Services, Cincinnati Children’s Hospital Research Center, Cincinnati, OH, USA** DNA Genotek, Ottawa, Ontario, Canada
2006-06-29

For long-range PCR, DNA from Oragene®/saliva samples
is equal in performance to DNA isolated from blood.

Roche expand long
PCR kitFinal concentration

Introduction

Buffer

1×Long-range PCR has a number of applications inhuman genetics including the detection of insertions
and deletions1 and SNP genotyping2, 3. To be
successful, long-range PCR requires DNA of highquality and high molecular weight such as thatobtained with the Oragene self‑collection kit, which
has a molecular weight greater than 23 kilobases4.
This study compares the long-range PCR performanceof DNA from Oragene/saliva samples purified with
prepIT™•L2P versus DNA purified from blood.

MgCl2

2.25 mM

dNTPs

500 µM

Forward primer

20 pmol

Reverse primer

20 pmol

Enzyme mix

1U

Template DNA

200 ngMaterials and methods
DNA purification
Saliva was collected using the Oragene self‑collection kit
and DNA was purified using the prepIT•L2P purification
protocol5. Additional DNA was isolated from blood andpurified using the MagneSil® Blood Genomic, Max Yield
System (Promega) on the KingFisher 96 automatedrobotic system (Thermo Electron).

Long-range PCR
A 7.3 kb fragment of the human GBA gene wasamplified using the Expand Long PCR kit with Buffer
System 2 (Roche Diagnostics). Primers specific to thehuman GBA gene were synthesized by Integrated
DNA Technologies Inc. (Table 1). All PCR reagentswere used at the final concentrations recommended
by the manufacturer (Table 2).

Table 2: Final PCR reagent concentrations.

Thermal cycling was performed using a DNA Engine
Tetrad machine (MJ Research) and the PCR programin Table 3. PCR products were loaded on 0.8% agarosegels and run for 1 hour and 20 minutes at 80 V. Afterelectrophoresis, gels were stained with ethidium
bromide and photographed.
Cycle

TemperatureTime

1×93°C

2 minutes

10×93°C

10 seconds

10×60°C

30 seconds

10×68°C

4 minutes 40 seconds

20×93°C

10 seconds

20×60°C

30 seconds

20×68°C

4 minutes 40 seconds+ 20 seconds per cycle

PrimerSequence1×68°C

7 minutes

GBA Forward

5’- TTC TCC ATG CAA ATC TGT GT-3’

1×4°C

HoldGBA Reverse

5’- GAA CCA GAT CCT ATC TGT GC-3’

Table 3: Thermal cycling parameters.

Table 1: Primer sequences.
†Saliva samples were collected with Oragene®•DNA or Oragene®•DISCOVER.

(outside the USA)(in the USA)

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ResultsDiscussion and conclusions

Figure 1 shows the results of long-range PCR using
DNA from Oragene/saliva samples and DNA fromblood. Lanes 1 to 3 show the results from Orageneand Lane 5 from blood. Both DNA types produced
the expected 7.3 kb amplicon from the human
GBA gene.

The results show that Oragene-purified DNA gives
equal performance to DNA isolated from blood.
The Oragene kit minimizes DNA shearing andgenerates high-molecular-weight DNA that is ideal
for long-range PCR applications.

Oragene
M123Blood
45

M

8 kb

Figure 1: Agarose gel of long-range PCR reactions. Lanes 1 to 3 used
DNA from Oragene/saliva samples and Lane 5 used DNA from blood.
Lane 4 is a negative control.

References
12345Dabora, S., Nieto, A., Franz, D., Jozwiak, S., Van Den Ouweland, A. and Kwiatkowski, D. (2000). Characterisation of six large
deletions in TSC2 identified using long-range PCR suggests diverse mechanisms including Alu mediated recombination.
Journal of Medical Genetics. 37(11), 877-883.
Nagano, M., Nakamura, T., Ozawa, S., Maekawa, K., Saito, Y. and Sawada, J. (2003). Allele-specific long-range PCR/sequencing
method for allelic assignment of multiple single nucleotide polymorphisms. Journal of Biochemical & Biophysical Methods. 55(1), 1-9.
Plaschke, J., Ruschoff, J. and Schackert, H. (2003). Genomic rearrangements of hMSH6 contribute to the genetic predispositionin suspected hereditary non-polyposis colorectal cancer syndrome. Journal of Medical Genetics. 40(8), 597-600.
Birnboim, H.C. (2004). Long-term stability of DNA from saliva samples stored in the Oragene self-collection kit.
DNA Genotek. PD‑WP-005.
Laboratory protocol for manual purification of DNA from 0.5 mL of sample. DNA Genotek. PD-PR-006.

Oragene®•DNA is not available for sale in the United States.
Oragene®•DISCOVER is for research use only, not for use in diagnostic procedures.
®Oragene is a registered trademark and prepIT™ is a trademark of DNA Genotek Inc. All other brands and names contained herein are the property of their respective owners.
All DNA Genotek protocols, white papers and application notes, are available in the support section of our website at www.dnagenotek.com.

MK-AN-013 Issue 3/2011-12© 2011 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved.

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