Different chemical methods used to attach oligonucleotides by their 5'-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5'-specific linkage that satisfyingly withstands PCR conditions and leaves the 3'-ends available for DNA polymerase activity. The best results were obtained with 5'-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5'-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.

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SYNOPSISUsing a sensitive birefringence instrument, the birefringence arising from the orientation of the DNA chain during electrophoretic transport has been recorded. This birefringence is shown to proceed both from the alignment (stretching) of the molecule in the direction of the electric field and from the extension of the length of its primitive path (overstretching) . The contribution of these two processes can be separated in the decay of the birefringence after the end of the application of the electric field. The fast relaxation of the overstretching occurs first and is demonstrated to be the main contribution to the birefringence. The orientation factor of the remaining stretched state and its decay can be quantitatively understood using the biased reptation model. It provides, in addition, a high value for the tube diameter or gel pore size a (4500 k 450 A for a 0.7% agarose gel with a c;'.~ dependence in the agarose concentration cg) and a low value for the effective charge per base pair (0.2e as compared to 0.5e using the condensation hypothesis). The contribution of overstretching to the birefringence is also quantitatively interpreted in term of the change in the mean length 1 of DNA inside a pore size a. The dynamics of decay of this overstretching is well represented by a stretched exponential with a stretching exponent a = 0.44. The mean decay time decreases slightly with increasing fields and scales with the overall DNA length close to N ; . 0 1993 John Wiley & Sons, Inc.