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DOP-PCR 315
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Degenerate Oligonucleotide-Primed PCR
Michaela Aubele and Jan Smida
1. Introduction
The amount of genomic DNA available for genetic studies can often be limiting.
Degenerated oligonucleotide-primed polymerase chain reaction (DOP-PCR) is an
appropriate method for overcoming these limitations by efficiently performing whole
genome amplification. The DOP-PCR technique is increasingly being applied for
simultaneous amplification of multiple loci in target DNA using oligonucleotide
primers of partially degenerate sequences (1). Contrary to other PCR-based general
amplification methods (Alu-PCR or IRS-PCR), DOP-PCR is a species-independent
technique for the amplification of small amounts of DNA.
Briefly, the use of one single degenerated primer allows random initial priming all
over the target DNA during the first five cycles of PCR at low annealing temperatures.
In subsequent cycles, more stringent conditions are then used to amplify the first PCR
products. DOP-PCR technique is recently being applied to produce sufficient amounts
of DNA from clinical tumor samples, in forensic analyses, prenatal diagnosis, and
many other investigations. Slightly different protocols have been used and optimized
worldwide. The protocol described here is adapted for use with microdissected,
formalin-fixed, paraffin-embedded human tissue in comparative genomic hybridization
analysis. A comparison of several DOP-PCR variations is also given by Larsen
et al. (2).
2. Materials
2.1. Deparaffination and Sampling of Material
1. Xylene (100%).
2. Ethanol (100%, 70%, 50%).
3. Distilled water.
4. Mayer’s Hemalaun solution (Merck 109 249, Darmstadt, Germany).
5. Laser buffer: 100 mM Tris-HCl, pH 7.5.
6. Proteinase K: 10 mg/mL proteinase K, pH 7.5 (Merck, Darmstadt, Germany).
7. Qiagen DNA Mini Kit (Qiagen, Hilden, Germany).
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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