ABSTRACT
Endonuclease VII (endo VII) binds to non-pairing nucleotides in DNA. This served as the basis for the development of a mutation detection assay involving immobilised endo VII and heteroduplex DNAs made by hybridisation of mutant and wild type DNA. The use of microtiter plates allows screening of large numbers of samples. Localisation of mutations in positive samples can be done in the same assay in a second optional step.
Mutations are inheritable changes in the sequence of the genetic material (mostly DNA) of an organism which can cause fatal defects like hereditary diseases or cancer (recent reviews in 1). Therefore, methods for mutation detection are gaining increasing importance especially in medical diagnostics. Mutations can be localised with great precision by DNA sequencing (2). The procedure is, however, time consuming, expensive, and requires toxic chemicals. Additional fast and low-cost alternatives have, therefore, been developed. In general these procedures measure mutations via mispairings in heteroduplex DNAs obtained after annealing wild type with mutant DNA in vitro (reviewed in 3). Besides physical and chemical methods, enzymatic assay systems using proteins involved in DNA repair have been established. One of these enzymes is endonuclease VII (endo VII) of bacteriophage T4 (phage T4) (4,5). Endo VII reports all possible mismatches including C/C, heteroduplex loops and single nucleotide bulges, single-strand overhangs, branched DNAs, bulky adducts, psoralen crosslinks and apurinic sites (6). The broad substrate specificity makes the enzyme an extremely versatile tool for mutation detection (7). The nucleolytic activity of endo VII has been used successfully to detect mutations in heteroduplex DNAs (8).
Here we describe an improved method that is based on the binding of mismatches and bulges in heteroduplex DNAs by immobilised endo VII in the absence of Mg2+. The use of microtiter plates allows screening of large numbers of samples making the procedure fast, easy and versatile. Localisation of mutations in the heteroduplex can still be done in the usual way by incubating aliquots of positive samples in the presence of Mg2+ (9,10). This step was applied successfully to re-check positives for false-positives and is strongly recommended for general use. For details see supplementary material online.
The binding assay was tested for all possible mismatches centrally located in heteroduplex DNAs of 84 and 164 bp using gel purified PCR-made strands for annealing. Heteroduplexes of 263 bp with a centrally located C/C mismatch or an 8 nt insertion made by the same procedure were also used.
For coating wells of microtiter plates, highly purified endo VII (4) was diluted to a final concentration of 20 µg/ml in Phosphate buffer containing 75 mM potassium phosphate buffer (pH 6.5) and 5 mM EDTA. Phosphate buffer was chosen at pH 6.5 instead of Tris-HCl buffer at pH 8.0 as used in earlier studies with phage T4 enzyme since the cloned enzyme shows considerably higher specific activity under these conditions (11). In a standard reaction procedure, 1 µg of endo VII was added in 50 µl to each well of a 96-well microtiter plate and incubated at room temperature for 30-60 min. Longer incubation times did not influence the results. The plates can be used immediately or stored in a humid atmosphere at 4°C for up to 7 days without loss of activity. The protein containing coating solution is not removed from the wells and sample DNA is added directly.
For binding ~3 fmol of radioactively labelled heteroduplex DNA were added to the protein solution in the wells of microtiter plates, mixed gently and then incubated for 2 h in a humid atmosphere at room temperature. Liquid was discarded and unbound DNA and protein were removed by washing the plates five times each with 200 µl of the incubation buffer per well. For release of bound DNA from the well, 50 µl of 1% SDS solution was added. Repeated pipetting ensured complete removal. For visualisation and documentation, the sample volume was kept small and reduced by vaporisation if necessary. An aliquot of 5-10 µl of liquid was spotted on small Whatman 3MM filters, air dried and quantitated with a phosphorimager (FujiBas 1000).Yes-or-no answers report presence or absence of mutations and large screens of heteroduplex samples can be processed in less than a few hours.
As shown in Figure 1 all mismatches were clearly detected. Differences among individual mismatches were recognised. The affinity of endo VII to a mixture of C/C and G/G mismatches was the highest, and to a mixture of A/A and T/T mismatches the lowest. The signal-to-noise ratio between mismatch and control was in all cases better than two (Table 1).
Nucleic Acids Research
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REFERENCES
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