Open AccessDNA‐DNA Hybridization on Nitrocellulose Filters
Author(s) -
FLAVELL Richard A.,
BORST Piet,
BIRFELDER E. Joyce
Publication year - 1974
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1974.tb03723.x
Subject(s) - dna , sequencing by hybridization , nucleic acid thermodynamics , nitrocellulose , dna–dna hybridization , duplex (building) , dna sequencing , microbiology and biotechnology , chemistry , biophysics , biology , biochemistry , base sequence , membrane , dna sequencer
1. Comparison of DNA‐DNA filter hybridization using either denaturated duplex DNA or artificial mixtures of the two complementary strands of T7 DNA shows that the competing renaturation reaction has two effects: (a) sequestering of 20–30% of the input DNA as duplex, unavailable for hybridization, and (b) hybridization of partially renatured DNA as a concatenane to the filter‐bound DNA. 2. Experiments using artificial mixtures of 32 P‐labelled and 3 H‐labelled L and H strands of T7 show that 10–20% of the total hybrid bound results from concatenation. With a 1:1 input ratio of complementary strands in solution, 10% of the input bound to filter DNA of a single strand ( e.g. L) is of the homologous strand ( i.e. L). This effect is relatively unaffected by the concentration of DNA in solution. 3. Variation of the L:H ratio in solution shows that the strand in excess hybridizes and renatures more efficiently. This effect gives an underestimation of the amount of the minor strand present in solution. The numerical values obtained here are likely to be strongly dependent on the complexity of the DNA and the precise hybridization conditions. 4. The implication of the results for homology experiments and determination of strand specificity in transcription is discussed.