Chapter 30
PROTOCOL
Microdissection of Chromosomes and Reverse FISH
ILSE CHUDOBA and GABRIELE SENGER
Introduction
Microdissection of chromosomes or chromosomal regions was first published by Scalenghe et al. in 1981. They dissected fragments from the polytene X chromosome of Drosophila melanogaster. The DNA was extracted
and ligated to a lambda vector. Already in this early work, it was demonstrated that the resulting clones can be hybridized back to the original
section of the X chromosome, where the dissection was carried out. However, application of this method to mammalian chromosomes was hampered due to the fact that normal metaphase chromosomes contain only
two double helices of DNA and hence a large number of fragments (more
than 100) had to be collected in order to obtain sufficient DNA for successful cloning in a lambda vector. A significant improvement of the original technique was achieved by the application of G-banding prior to the
dissection procedure (Senger et ai. 1990) and by establishing an efficient
micro-cloning system (Liidecke et al. 1989). However, the real breakthrough for the microdissection technique was the invention of the sequence-independent DNA amplification. Different protocols have been
shown to be efficient in amplifying a DNA pool with no specific priming
sites (Bohlander et al. 1992, Telenius et al. 1992). However, it turns out that
the polymerase chain reaction using a degenerate oligonucleotide primer
(DOP-PCR) (Telenius et al. 1992) is the preferred amplification technique
which is applied in most experiments. The original technique of Telenius
was adapted in different ways (Meltzer et ai. 1992, Viersbach et ai. 1994,
Miiller-Navia et ai. 1995, Rubtsov et al. 1996, Chudoba et aI., 1996). Regardless of the follow-up applications, such as positional cloning or fluor~ Ilse Chudoba, MetaSystems GmbH, Robert-Bosch-Str. 6, Alt1ussheim, 68804,
Germany (phone +49-6205-39610; fax +49-6205-32270; e-mail ic@metasystems.de)
Gabriele Senger, Praxis fUr Medizinische Genetik, RoritzerstraBe 2, Regensburg, 93047,
Germany
PROTOCOL
Microdissection of Chromosomes and Reverse FISH
ILSE CHUDOBA and GABRIELE SENGER
Introduction
Microdissection of chromosomes or chromosomal regions was first published by Scalenghe et al. in 1981. They dissected fragments from the polytene X chromosome of Drosophila melanogaster. The DNA was extracted
and ligated to a lambda vector. Already in this early work, it was demonstrated that the resulting clones can be hybridized back to the original
section of the X chromosome, where the dissection was carried out. However, application of this method to mammalian chromosomes was hampered due to the fact that normal metaphase chromosomes contain only
two double helices of DNA and hence a large number of fragments (more
than 100) had to be collected in order to obtain sufficient DNA for successful cloning in a lambda vector. A significant improvement of the original technique was achieved by the application of G-banding prior to the
dissection procedure (Senger et ai. 1990) and by establishing an efficient
micro-cloning system (Liidecke et al. 1989). However, the real breakthrough for the microdissection technique was the invention of the sequence-independent DNA amplification. Different protocols have been
shown to be efficient in amplifying a DNA pool with no specific priming
sites (Bohlander et al. 1992, Telenius et al. 1992). However, it turns out that
the polymerase chain reaction using a degenerate oligonucleotide primer
(DOP-PCR) (Telenius et al. 1992) is the preferred amplification technique
which is applied in most experiments. The original technique of Telenius
was adapted in different ways (Meltzer et ai. 1992, Viersbach et ai. 1994,
Miiller-Navia et ai. 1995, Rubtsov et al. 1996, Chudoba et aI., 1996). Regardless of the follow-up applications, such as positional cloning or fluor~ Ilse Chudoba, MetaSystems GmbH, Robert-Bosch-Str. 6, Alt1ussheim, 68804,
Germany (phone +49-6205-39610; fax +49-6205-32270; e-mail ic@metasystems.de)
Gabriele Senger, Praxis fUr Medizinische Genetik, RoritzerstraBe 2, Regensburg, 93047,
Germany