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Paul Burr, Wellcome Clinical Scholar, Department of Veterinary Pathology, University of Glasgow, Bearsden Rd, Glasgow. G61 1QH.
Introduction
A method that can be conveniently used to prepare DNA from a wide range
of tissues for analysis in the Polymerase Chain Reaction is described.
The method is both quick and simple, with a low risk of inadvertent contamination
of samples due to the reduced number of manipulations required compared
to other DNA extraction methods.
Methods
Canine tissues (tri-geminal ganglion, lumbo-sacral ganglion, brain, mesenteric
lymph node, sub-mandibular lymph node, tonsil, sub-mandibular salivary
gland, parotid salivary gland, spleen, liver and kidney) were collected
from a number of dogs during routine post mortem. All tissues were collected
within 2 hours of death and frozen at -70 C until processed. Great care
was taken during preparation to avoid problems of both environmental and
cross contamination.
1. Cut a small (3 mm3) piece of tissue on a fresh glass slide using a fresh scalpel blade. Macerate slightly with the blade and place in a sterile 1.5 ml screw cap Eppendorf tube.
2. Add 1 ml of 1x Trypsin/EDTA (Gibco BRL). Incubate at room temperature for 15-30 minutes, shaking occasionally.
3. Spin down (13K, 2-3 minutes) to pellet tissue, and carefully remove all the supernatant.
4. Add 250 l of InstaGene matrix (Bio-Rad Laboratories), and mix well. Incubate at 56 C for 30 minutes, mixing occasionally.
5. Vortex tube thoroughly for 10 seconds, place in boiling water bath or 100 C heat block for 8 minutes.
6. Vortex thoroughly for 10 seconds, spin at 13K for 2-3 minutes. Use 20 l of the supernatant for each PCR reaction.
Notes: Store remainder at -20 C for subsequent PCRs. Repeat step 6 when reusing the preparation (allow to thaw first).
PCR
Primers used in this experiment were designed to amplify a 120 base pair
fragment of the Canine Pancreatic Lipase Gene (Mickel et al., 1989). Amplification
was carried out using 2 Units AmpliTaq DNA polymerase (Perkin-Elmer) mixed
with an equal volume of TaqStart antibody (Clontech) per reaction in a
total reaction volume of 50 l, containing 50 pmol each primer and 200
mol/L of each deoxynucleoside triphosphate in 10 mM Tris (pH 8.4), 50
mM KCl, and 1.5 mM MgCl2. Thermal cycling was performed on a Perkin-Elmer
9600. Initial denaturation was at 94 C for 5 minutes, followed by 40
cycles of 94 C for 30 seconds, 57.5 C for 1 minute, then a final extension
of 72 C for 7 minutes.
Results
All tissues examined produced a clear band of the correct size. The variation in the brightness of the band does not appear to correspond to the amount of DNA template present in each reaction. Apart from the positive control (A72 cell line), there are two template copies per cell for each tissue. The most likely cause of the variation in intensity of the band obtained is therefore inhibitory factors present within the sample. It is important not to exceed the capacity of the InstaGene matrix by using too much tissue in the initial preparation. Exceeding the matrix capacity may cause PCR-inhibitory factors normally removed by the procedure to remain in the supernatant. Carryover of these inhibitory factors may affect amplification in the subsequent PCR reaction.
Conclusion
InstaGene matrix can be used to rapidly prepare PCR compatible DNA from
a wide range of tissues in about 2 hours. Preparation and amplification
can be carried out in the same day if required.
Acknowledgements
This work was supported by a grant from The Wellcome Trust.
References
Binn, L. N., Marchwicki, R. H. and Stephenson, E. H., Establishment of
a Canine Cell Line: Derivation, Characterization, and Viral Spectrum,
Am. J. Vet. Res. 41, 855-860 (1980).
Mickel, F. S., Weidenbach, F., Swarovsky, B., Laforge, K. S. and Scheele, G. A., Structure of the Canine Pancreatic Lipase Gene, J.Biol.Chem., 264, 12895-12901 (1989).
* The Polymerase Chain Reaction (PCR) process is covered by patents owned
by Hoffmann-LaRoche. Use of the PCR process requires a license.
Eppendorf is a trademark of Eppendorf Geratebau netheler + Hinz, GmbH.
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