Combination of amplification and post-amplification strategies to improve optical DNA sensing.
The work evaluated a series of approaches to optimise detection of polymerase chain reaction (PCR) amplified DNA samples by an optical sensor based on surface plasmon resonance (SPR) (BiacoreX). The optimised procedure was based on an asymmetric PCR amplification system to amplify predominantly one DNA strand, containing the sequence complementary to a specific probe. The study moved into two directions, aiming to improve the analytical performance of SPR detection in PCR amplified products. One approach concerned the application of new strategies at the level of PCR, i.e. asymmetric PCR to obtain ssDNA amplified fragments containing the target capable of hybridisation with the immobilised complementary probe. The other strategy focused on the post-PCR amplification stage. Optimised denaturing conditions were applied to both symmetrically and asymmetrically amplified fragments. The effective combination of the two strategies allowed a rapid and specific hybridisation reaction. The developed method was successfully applied in the detection of genetically modified organisms.
Giakoumaki
Elsa
E
Cranfield Biotechnology Centre, Cranfield University, Silsoe, Bedfordshire MK45 4DT, UK.
Minunni
Maria
M
Tombelli
Sara
S
Tothill
Ibtisam E
IE
Mascini
Marco
M
Bogani
Patrizia
P
Buiatti
Marcello
M
D015374:Biosensing Techniques / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000235:genetics; D015342:DNA Probes / Q000138:chemical synthesis / Q000737:chemistry*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D009691:Nucleic Acid Denaturation; D055095:Optics and Photonics; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*
D015342:DNA Probes; D004247:DNA