John M. S. Bartlett.pdf - Bio-Nica.info

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.