Chapter 10
Hybridization Methods
(Southern and Northern Blotting)
KAI-OLAF NETZER
lntroduction
Hybridization methods represent standard techniques in molecular biology. In general, they are used to detect particular sequences (target) within
a complex mixture of DNA or RNA molecules. DNA or RNA are usually
transferred and immobilized to nitrocellulose or, more commonly, to nylon
membranes. Complementary single-stranded probes are labeled radioactively or non-radioactively. When hybridized to the filter, probes bind to
their complementary target sequence via hydrogen bonds. Unhybridized
probe is then washed away, and specifically-bound probe is detected by
autoradiography or color reaction.
Hydrogen bonds produce a stable but reversible association of doublestranded DNA. For replication and transcription in vivo, it is most important that the two DNA strands can be separated and reassociated under
physiological conditions without the need to break covalent bonds. This
natural function represents the basis for the in vitro techniques described
in this section. The term hybridization refers to the association of complementary single-stranded DNA or RNA molecules to produce hybrid doublestranded molecules.
Hybridization can occur
• in solution with the participating nucleic acid molecules being present as
single strands,
• with one strand (target) immobilized to a filter membrane, and the other
strand (probe) in solution, and
• in situ, with the target strand fixed in tissue sections, cells, or chromosome smears, and the probe present in solution.
Kai-OlafNetzer, Krankenhaus Köln-Merheim, Medizinische Klinik I, Ostmerheimer Str.
200, Köln, 51109, Germany (phone +49-221-8907-2200; fax +49-221-8907-2335; e-mail
kai.netzer@uni-koeln.de)
Hybridization Methods
(Southern and Northern Blotting)
KAI-OLAF NETZER
lntroduction
Hybridization methods represent standard techniques in molecular biology. In general, they are used to detect particular sequences (target) within
a complex mixture of DNA or RNA molecules. DNA or RNA are usually
transferred and immobilized to nitrocellulose or, more commonly, to nylon
membranes. Complementary single-stranded probes are labeled radioactively or non-radioactively. When hybridized to the filter, probes bind to
their complementary target sequence via hydrogen bonds. Unhybridized
probe is then washed away, and specifically-bound probe is detected by
autoradiography or color reaction.
Hydrogen bonds produce a stable but reversible association of doublestranded DNA. For replication and transcription in vivo, it is most important that the two DNA strands can be separated and reassociated under
physiological conditions without the need to break covalent bonds. This
natural function represents the basis for the in vitro techniques described
in this section. The term hybridization refers to the association of complementary single-stranded DNA or RNA molecules to produce hybrid doublestranded molecules.
Hybridization can occur
• in solution with the participating nucleic acid molecules being present as
single strands,
• with one strand (target) immobilized to a filter membrane, and the other
strand (probe) in solution, and
• in situ, with the target strand fixed in tissue sections, cells, or chromosome smears, and the probe present in solution.
Kai-OlafNetzer, Krankenhaus Köln-Merheim, Medizinische Klinik I, Ostmerheimer Str.
200, Köln, 51109, Germany (phone +49-221-8907-2200; fax +49-221-8907-2335; e-mail
kai.netzer@uni-koeln.de)