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VOLKER JUNG and GEORG A. WEBER
to provide enough DNA for standard CGH. With an universal PCR using
degenerated oligonucleotides primers (DOP-PCR) and low stringency cycle conditions (Telenius et aI., 1992, Miiller-Navia et al., 1995, Kuukasjarvi
et aI., 1997), it is possible to uniformly amplify very small microdissected
DNA samples.
Primers used for DOP-PCR (see Fig. 2) contain a variable binding region with a sequence of six randomly combined nucleotides which is
flanked by a stringent primer binding region at the 5-end (ten nucleotides)
and a main binding region at the 3-end (six nucleotides). The amplification procedure is divided into two steps. During the first step, eight PCR
cycles are performed at low stringency conditions (annealing temperature
30°C), followed by a slow temperature increase to the elongation temperature, and primer elongation.
During the second step (30-35 cycles) a more stringent (higher) annealing temperature (Ta=62°C) is used, and all DNA sequences generated during the first eight cycles are exponentially amplified. DOP-PCR amplifications are useful for CGH studies and result in a smear of DNA fragments
which is visible on an agarose minigel stained with ethidium bromide.
Two alternative protocols exist for an efficient amplification of the
small isolated DNA samples for CGH studies.
The standard protocol combines the two amplification steps in one
PCR reaction with the same Taq polymerase (Speicher et aI., 1993, Isola
et aI., 1994, Wiltshire et al., 1995).
The other protocol (Kuukasjarvi et al., 1997) separates the DOP-PCR
into two different reactions: The first unspecific step called preamplification is performed with ThermoSequenase followed by adding a Taq polymerase for the second amplification step. In our experience the standard
protocol produced sufficient probes for CGH studies with a homogeneous
staining pattern of all chromosomes. The probes which were generated
with ThermoSequenase seem to be less suitable for CGH studies because
the hybridization results in an inhomogeneous staining pattern that is
very difficult to interpret.
The quality of the DNA which is isolated from paraffin-embedded tissue depends on two important parameters: First, the use of 4% neutral
buffered formaldehyde for the fixation; second, duration of the fixation
should be no longer than 1 day.
VOLKER JUNG and GEORG A. WEBER
to provide enough DNA for standard CGH. With an universal PCR using
degenerated oligonucleotides primers (DOP-PCR) and low stringency cycle conditions (Telenius et aI., 1992, Miiller-Navia et al., 1995, Kuukasjarvi
et aI., 1997), it is possible to uniformly amplify very small microdissected
DNA samples.
Primers used for DOP-PCR (see Fig. 2) contain a variable binding region with a sequence of six randomly combined nucleotides which is
flanked by a stringent primer binding region at the 5-end (ten nucleotides)
and a main binding region at the 3-end (six nucleotides). The amplification procedure is divided into two steps. During the first step, eight PCR
cycles are performed at low stringency conditions (annealing temperature
30°C), followed by a slow temperature increase to the elongation temperature, and primer elongation.
During the second step (30-35 cycles) a more stringent (higher) annealing temperature (Ta=62°C) is used, and all DNA sequences generated during the first eight cycles are exponentially amplified. DOP-PCR amplifications are useful for CGH studies and result in a smear of DNA fragments
which is visible on an agarose minigel stained with ethidium bromide.
Two alternative protocols exist for an efficient amplification of the
small isolated DNA samples for CGH studies.
The standard protocol combines the two amplification steps in one
PCR reaction with the same Taq polymerase (Speicher et aI., 1993, Isola
et aI., 1994, Wiltshire et al., 1995).
The other protocol (Kuukasjarvi et al., 1997) separates the DOP-PCR
into two different reactions: The first unspecific step called preamplification is performed with ThermoSequenase followed by adding a Taq polymerase for the second amplification step. In our experience the standard
protocol produced sufficient probes for CGH studies with a homogeneous
staining pattern of all chromosomes. The probes which were generated
with ThermoSequenase seem to be less suitable for CGH studies because
the hybridization results in an inhomogeneous staining pattern that is
very difficult to interpret.
The quality of the DNA which is isolated from paraffin-embedded tissue depends on two important parameters: First, the use of 4% neutral
buffered formaldehyde for the fixation; second, duration of the fixation
should be no longer than 1 day.