Chapter 15
Preparation of Samples for CGH by DOP-PCR from
Cryofixed or Paraffin-Embedded, Microdissected Cells
A comparison of two different PCR amplification methods
VOLKER JUNG and GEORG A. WEBER
Introduction
The development of the "comparative genomic hybridization (CGH)"
technique (Kallioniemi et aI., 1992) enabled the cytogeneticist to comprehensively analyze chromosomal imbalances in entire genomes. The technique reveals chromosomal regions with relative gains or losses of DNA
sequences, by comparing DNA from a test specimen (test DNA) with the
DNA from a normal specimen (reference DNA). Test DNA labeled e.g.
with biotin is mixed 1:1 with differently labeled reference DNA with digoxigenin and simultaneously hybridized to normal metaphase spreads.
For the detection of the hybridized test and reference DNA, different
fluorochromes are used, and each is visualized by epifluorescence microscopy with selective fllters. Regions within the tumor that are over- or
underrepresented in the probe mixture can be identified by an increased
or decreased color ratio of the two fluorochromes.
The standard CGH protocol requires 0.5-1.0 Ilg of genomic DNA, corresponding to approximately 50,000 to 100,000 diploid cells.
Many questions in pathology or in tumor genetics cannot be answered
because of the lack of a sufficient amount of fresh material. In many cases,
paraffin-embedded specimens are the only available tumor material. Microdissection of different tumor areas of a given tumor from the same
tissue section (cryoflXed or paraffin-embedded) is an elegant method
to investigate focal heterogeneity within the specimen (Aubele et aI.,
1999, Jung et al., 1999). Microdissected probes, however, are too small
~ Volker Jung, Saarland University, Institute of Human Genetics, Building 68,
Homburg/Saar, 66421, Germany (phone +49-6841-1626605; fax +49-6841-1626600;
e-mail volker.jung@uniklinik-saarland.de)
Georg A. Weber, Saarland University, Institute of Human Genetics, Building 68,
Homburg/Saar, 66421, Germany (phone +49-6841-1626605; fax +49-6841-1626600;
e-mail georg.weber@uniklinik-saarland.de)
PROTOCOL
Preparation of Samples for CGH by DOP-PCR from
Cryofixed or Paraffin-Embedded, Microdissected Cells
A comparison of two different PCR amplification methods
VOLKER JUNG and GEORG A. WEBER
Introduction
The development of the "comparative genomic hybridization (CGH)"
technique (Kallioniemi et aI., 1992) enabled the cytogeneticist to comprehensively analyze chromosomal imbalances in entire genomes. The technique reveals chromosomal regions with relative gains or losses of DNA
sequences, by comparing DNA from a test specimen (test DNA) with the
DNA from a normal specimen (reference DNA). Test DNA labeled e.g.
with biotin is mixed 1:1 with differently labeled reference DNA with digoxigenin and simultaneously hybridized to normal metaphase spreads.
For the detection of the hybridized test and reference DNA, different
fluorochromes are used, and each is visualized by epifluorescence microscopy with selective fllters. Regions within the tumor that are over- or
underrepresented in the probe mixture can be identified by an increased
or decreased color ratio of the two fluorochromes.
The standard CGH protocol requires 0.5-1.0 Ilg of genomic DNA, corresponding to approximately 50,000 to 100,000 diploid cells.
Many questions in pathology or in tumor genetics cannot be answered
because of the lack of a sufficient amount of fresh material. In many cases,
paraffin-embedded specimens are the only available tumor material. Microdissection of different tumor areas of a given tumor from the same
tissue section (cryoflXed or paraffin-embedded) is an elegant method
to investigate focal heterogeneity within the specimen (Aubele et aI.,
1999, Jung et al., 1999). Microdissected probes, however, are too small
~ Volker Jung, Saarland University, Institute of Human Genetics, Building 68,
Homburg/Saar, 66421, Germany (phone +49-6841-1626605; fax +49-6841-1626600;
e-mail volker.jung@uniklinik-saarland.de)
Georg A. Weber, Saarland University, Institute of Human Genetics, Building 68,
Homburg/Saar, 66421, Germany (phone +49-6841-1626605; fax +49-6841-1626600;
e-mail georg.weber@uniklinik-saarland.de)
PROTOCOL