DNA blots

Fig. 11.13. DNA blot analysis using spots from chromosomes sorted directly onto filter paper on the basis of their Hoechst 33258 fluorescence. From Van Dilla et al. (1990).

The accurate measurement of the concentration of DNA appeared to be essential because of the strong dependence of the chemiluminescence signal upon the amount of DNA blotted on the filter. Approximately, a 2-fold increase in the amount of DNA resulted in a 4-fold increase of the chemiluminescence signal. For that reason we decided to blot, as a standard procedure, 1 pg DNA/blot instead of various amounts of DNA and to reserve 10 positions on the 96-blots filter for calibration samples of DNA with adduct levels in the range of 0-10 N7-HETE-dG/107 nucleotides. 

The term Southern blotting is used because the method was developed by E. M. Southern and the term northern blotting was introduced for RNA blots to distinguish them from DNA blots. More recently, protein chemists have gotten into the act by blotting electrophoresed proteins to a membrane and detecting specific protein molecules with antibodies, which is known as western blotting. Finally, southwestern blots (a renatured protein blot and a DNA probe) are used to study protein-DNA interactions. 

Khandjian, E. W. (1987) Optimized hybridization of DNA blotted and fixed to nitrocellulose and nylon membranes. Biotechnology 5, 165-167. 

Fig. 1. Single copy gene detection. 1, 2, and S pg of an coRI digest of human genomic DNA blotted onto Hybond-N. Hybridized with a 1.5-kb probe for N-ras proto-oncogene labeled with fluorescein-dUTP by the random primer labeling reaction see Chapter 15). Hybridization with 10 ng/mL of probe, overnight, at 60 C 30-min exposure on Hyperfilm-MP.

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