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DOP-PCR 317 Fig. 1. DOP-PCR products reveal a typical smear ranging from about 100 to 2500 bp. Lane 1: marker pBR 322, HaeIII; lane 2: positive control (PCR product with template DNA and a gene-specific primer (β-Actin); lane 3: negative control (PCR product without template and with degenerate primer); lane 4: empty; lanes 5 to 9: DNA samples from formalin-fixed, paraffin-embedded tissue that were amplified by DOP-PCR. 4. Notes 1. As in all preparations that require handling of small cell and/or small DNA amounts, the PCR mix should be set up in a laminar flow to avoid any contamination. Furthermore, because of the species-independent primer, a strict contamination-free working is prerequisite. 2. Tumor samples may be pretreated for 30 min with 2 units Topoisomerase I at 37°C to relax the template DNAs. Stop activation of topoisomerase at 90°C for 10 min and chill on ice, then add Taq DNA Polymerase and start PCR. This additional step is recommended for isolated fresh material but is not necessary for degraded DNA, as is the case for formalin-fixed, paraffin-embedded tissue. 3. All PCRs should be controlled for possible contamination by using at least one negative control (vial without template DNA), and one positive control (vial with template DNA and a gene specific primer, e.g., β-actin; Fig. 1). 4. The most commonly used DOP-PCR primer is 6-MW, originally described by Telenius (1), a degenerate primer with 6 specific 3′ bases and a XhoI site at its 5′ end (the same primer has been described as UW4B, UN-1, or MTE1 by different authors). 5. More than 40 cycles in DOP-PCR should be avoided. Based on the authors’ experiences, artificial results could arise. For this reason, labeling of DNA should be performed, for example, by Nick translation instead of a further DOP-PCR.
318 Aubele and Smida References 1. Telenius, H., Carter, N. C., Bebb, C. E., Nordenskjöld, M., Ponder, B. A. J, and Tunnacliffe, A. (1992) Degenerate oligonucleotide-primed PCR: General amplification of target DNA by a single degenerate primer. Genomics 13, 718–725. 2. Larsen, J., Ottesen, A. M., Lundsteen, C., Leffers, H., and Larsen, J. K. (2001) Optimization of DOP-PCR amplification of DNA for high resolution comparative genomic hybridization analysis. Cytometry 44, 317–325.
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63. Primed In Situ Nucleic Acid Lab
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44 Pearson 7. Add 60 µL of chlorof
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106 Wang and Young full-length cDNA
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156 Olmos et al. Table 2 Volume and
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158 Olmos et al. 8. The sensitivity
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160 Olmos et al.
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162 Abe 5. Moloney murine leukemia
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166 Abe 4. For HBV, nested PCR usin
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168 Tellier et al. of Pfu (and ther
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