Southern transfer

It is interesting to note that while column chromatography and centrifugation were developed for biomolecule purification and separation, many of the early diagnostic substrates for nucleic acids and proteins were membranes. For the Southern transfer process, the membrane provided a convenient way to interrogate sequences in genomic DNA fragments (Southern, 1975). 

Transfer the DNA probes to nitrocellulose filter by Southern transfer (23) Small fragments normally transfer within 4-6 h. 

After self-splicing of group I introns in vitro with [a-32P]GTP, the 5 end of the excised intron will be end labeled (see previous section). The intron can therefore be used as a radioactive RNA probe to detect homologous DNA sequences. The DNA under question is subjected to restriction digestion, fractionated on agarose gels, and bound to nitrocellulose or nylon filters by Southern transfer according to standard protocols (e.g., Ausubel et a/.13). Hybridization of the filter-bound DNA to the intron probe is carried out as follows. 

After Southern transfer of restriction-digested genomic DNA, filters are prehybridized for 1-4 hr. The probe is boiled for 10 min to denature it, added to fresh hybridization buffer, and hybridized overnight. To calculate appropriate hybridization temperatures in buffers with or without forma-mide, and for details of hybridization buffers and washes, see standard protocols.13 For hybridizations at low stringency to detect signals from fragments of approximately 65-70% overall sequence similarity, we apply 55° for hybridization and washes in 6X SSC without formamide. Similar protocols can be used for colony and plaque hybridizations. 

The number of rRNA operons in archaea is always small. Haloferax vol-canii, Methanobacterium thermoautotrophicum and Methanothermus fervidus have two, and Methanobacterium vannielii has four (reviewed by Brown et al. [5]). On the basis of Southern transfers of pulsed field gels, Sanz et al. [106] report that the halophilic archaea have from one to four rRNA operons Haloarcula californiae, 4 Haloferax gibbonsii, 4 Halobacterium halobium NCMB 111, 3 Halobacterium marismortui, 3 Halococcus morrhuae, 2 and Halobacterium salinarium, 1. Bacteria have from one to eleven copies (e.g.. Bacillus subtilis, 11 [107] Escherichia coli, 7 [108], and Mycoplasma pneumoniae, 1 [109]). In Halobacterium halobium, the presence of only one rRNA operon facilitated the isolation of strains with mutated 23S rRNA genes, which are resistant to thiostrepton [110], anisomycin [111] or chloramphenicol [112]. 

Reproducibility of electrophoresis and Southern blotting is essential and can be maintained by careful quality control tests of the pH and conductivity of key reagents (e.g. electrophoresis buffer and SSC). The quality of fingerprints will be affected by poor Southern transfer of DNA. Therefore, transfer should always be checked by restaining blotted gels in electrophoresis buffer plus ethidium bromide (0.5 mg H) for 30 min. Inspection of the restained gel on a UV-transil-luminator should reveal no signs of residual DNA molecular weight markers. 

Two routine methods of preparing blots are employed depending upon the membrane chosen Southern transfer must be used for nitrocellulose and can be used for all nylon varieties. Alternatively, some nylon membranes are amenable to alkali transfer 1. 

Fig. 2. Restriction enzyme analysis and Southern-blot hybridization of human tumor DNA. Human tumor DNA (20 p-g) was isolated (16) and digested with coRI restriction enzyme, electrophoresed on a 0.8% agarose gel (A shows the ethidium-bromide stain) and after Southern transfer, probed with P-labeled pcD-p(ADP-ribose) polymerase cDNA, (B). Lanes 1, Human placental DNA 2, Laryngeal squamous ceU carcinoma (SQ-20B) 3, Lung adenocarcinoma (A549) 4, Cervical carcinoma (HeLaSs) Ewing s sarcoma (A4573) 6, normal human fibroblasts (NHF). Marker-lambda-DNA.

The southern transfer procedure has eilso been extended to RNA now. The nzime given to such transfer procedure is Northern blotting. There are some differences in the methodology between Southern and Northern procedures. The first major difference is that RNA is not denatured by alkali because it becomes hydrolysed with such treatment. Instead formaldehyde is used for the purpose. Secondly, RNA does not bind to nitrocellulose unless denatured. Thus, in Northern blotting diazoben lojgrmethyl (DBM) paper is used. This paper binds both RNA and DNA. 

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