Probing strategies for duplex DNA

• Main Author: Drewe, L. J.
• Published: University of Cambridge 2000
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598653

Two main approaches have been adopted for the specific detection of the duplex PCR amplicon. The first investigates the potential of denaturing the amplicon so that the resulting single-strand DNA can be applied to the biosensor interface derived with a DNA probe. The work in this thesis validated such an approach using a model single-strand DNA target-probe system and demonstrated that the extent of hybridisation could be improved 3.5-fold by replacing the immobilised DNA with a peptide nucleic acid (PNA) probe of the same sequence. Hybridisation was not, however, demonstrated when the analyte consisted of denatured duplex DNA. The second approach investigated a novel method for detecting duplex PCR amplicons without their prior denaturation. By exploiting the charge properties of PNA this nucleic acid may be manipulated to strand invade duplex DNA at polypurine stretches within the target molecule. A method for the universal incorporation of polypurine motifs into any amplicon using a polypyrimidine rich 5'-tail on the specific PCR primer was adopted to both confer and control the location of strand invasion sites. PNA strand invasion of PCR amplicons generated in this manner was demonstrated both in solution and on the sensor surface and was generally observed within 10 minutes. The relative merits of adopting such an approach for the sensitive, rapid and specific detection and identification of microorganisms is critically discussed in context with alternative methods for detecting microorganisms and further DNA hybridisation formats.