10 Hybridization Methods (Southern and Northern Blotting)
131
5. Layer the fllter membrane briefly on a sheet of dry filter paper (Whatman
3MM) to blot dry. Rinse in 5x SSC for 5 minutes.
6. Fix the DNA to the membrane. Nitrocellulose should be baked for 2
hours at 80°C under vacuum. Nylonfilters should be wrapped in SaranWrap( or any comparable plastic wrap while still wet, placed DNA side
down on a UV transilluminator and exposed to UV light (280 nm) for 3-5
minutes. The filter is then ready for hybridization.
Note: If not hybridized immediately, nitrocellulose filters should be stored
under vacuum. Nylon filters should be sealed in plastic bags and stored at
4°C.
Subprotocol 2
Preparation of Southern Blots for Genomic DNA
The preparation of Southern blots involves DNA digestion with restriction
enzymes, size fractionation by agarose gel electrophoresis, and transfer
from the agarose gel to a filter membrane by capillary flow of buffer
with high ionic strength, i.e. high sodium content (20x SSC). The technique
was described by Southern {1975), hence the name Southern blot. (With
reference to the Southern transfer ofDNA, transfer ofRNA to a filter membrane has been named Northern blot, and transfer of proteins to a filter
membrane has been named Western blot.) Transfer of DNA from gel to
filter membrane can be accelerated by the use of vacuum or positive pressure blot apparatuses which are commercially available from several suppliers.
Southern blots are used, for instance, to study genetic diseases. Genomic Applications
DNA for the analysis ofthe human genome can be extracted from any nucleated cell of an organism. Once blots are prepared from size-fractionated
DNA samples, the presence of abnormal sequences or the absence of normal
nucleic acid sequences can be verified by hybridization to specific probes.
Bands of altered size, or missing bands, may indicate structural aberrations
of a gene. On the other hand, variation in the size of restriction fragments
may as well be caused by genetic polymorphisms ( e.g. point mutations in
the recognition sequence of restriction enzymes) that do not necessarily
have a pathogenic effect. Polymorphisms are inherited according to mendelian principles. They result in restriction fragment length polymorphisms
(RFLP) that can be utilized as markers for specific sites within the genome.
Hence they can be used for linkage analyses (see Chap. 23).
131
5. Layer the fllter membrane briefly on a sheet of dry filter paper (Whatman
3MM) to blot dry. Rinse in 5x SSC for 5 minutes.
6. Fix the DNA to the membrane. Nitrocellulose should be baked for 2
hours at 80°C under vacuum. Nylonfilters should be wrapped in SaranWrap( or any comparable plastic wrap while still wet, placed DNA side
down on a UV transilluminator and exposed to UV light (280 nm) for 3-5
minutes. The filter is then ready for hybridization.
Note: If not hybridized immediately, nitrocellulose filters should be stored
under vacuum. Nylon filters should be sealed in plastic bags and stored at
4°C.
Subprotocol 2
Preparation of Southern Blots for Genomic DNA
The preparation of Southern blots involves DNA digestion with restriction
enzymes, size fractionation by agarose gel electrophoresis, and transfer
from the agarose gel to a filter membrane by capillary flow of buffer
with high ionic strength, i.e. high sodium content (20x SSC). The technique
was described by Southern {1975), hence the name Southern blot. (With
reference to the Southern transfer ofDNA, transfer ofRNA to a filter membrane has been named Northern blot, and transfer of proteins to a filter
membrane has been named Western blot.) Transfer of DNA from gel to
filter membrane can be accelerated by the use of vacuum or positive pressure blot apparatuses which are commercially available from several suppliers.
Southern blots are used, for instance, to study genetic diseases. Genomic Applications
DNA for the analysis ofthe human genome can be extracted from any nucleated cell of an organism. Once blots are prepared from size-fractionated
DNA samples, the presence of abnormal sequences or the absence of normal
nucleic acid sequences can be verified by hybridization to specific probes.
Bands of altered size, or missing bands, may indicate structural aberrations
of a gene. On the other hand, variation in the size of restriction fragments
may as well be caused by genetic polymorphisms ( e.g. point mutations in
the recognition sequence of restriction enzymes) that do not necessarily
have a pathogenic effect. Polymorphisms are inherited according to mendelian principles. They result in restriction fragment length polymorphisms
(RFLP) that can be utilized as markers for specific sites within the genome.
Hence they can be used for linkage analyses (see Chap. 23).