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Chapter 4
4.2.2 DNA recognition by PNA probes in solution
Probe performances were initially tested in solution by measuring the melting temperatures of
both fullmatch and mismatch PNA-DNA duplexes, by using oligonucleotides homologous to
the ApoE sequences (Table 4-1). Melting temperatures were evaluated as the first derivatives
of the UV absorption curves at 260 nm in a temperature range from 20 to 90°C.
The chiral PNA probes were found to bind with very high stability to complementary DNA
sequences (Tm from 69 to 85°C) and also exerted very high sequence recognition, with
differences between full match and mismatch melting temperatures ranging from 15°C to
19°C. The high stability of the full match complexes, together with the increased specificity,
make feasible to find conditions, after PNA deposition on a microarray, in which it is possible
to have good signals for full match duplexes and no signals for the mismatched ones.
4.2.3 PNA spotting on the array platform
Water SDS 0.001%
PNA Wash Wash Buffer
Water SDS 3%
PNA Wash Wash Buffer
Water SDS 5%
PNA Wash Wash Buffer
Water-CH 3 CN SDS 3%
Acetonitrile
Mixed
PNA
Wash
Wash
Buffer
PNA Wash Wash Buffer
PNA CH 3CN SDS 3% CH 3CN Buffer Buffer
Water :DMF 9/1
PNA Wash Wash Buffer
Carbonate Buffer pH=9 SDS 0.001%
PNA Wash Wash Wash Buffer
Figure 4-3: Optimization of washing procedure (effect of solution composition)
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