Author: Huang, Jie
Title: Modification of hybridization probes to resist cleavage by the 5' nuclease activity of Taq DNA polymerase
Degree: M.Sc.
Year: 2009
Subject: Hong Kong Polytechnic University -- Dissertations.
Polymerase chain reaction.
Molecular biology -- Methodology.
Department: Department of Health Technology and Informatics
Pages: xiii, 72 leaves : ill. (some col.) ; 30 cm.
Language: English
Abstract: The polymerase chain reaction (PCR) is a revolutionary technology for the amplification and detection of DNA and RNA. A few molecules of DNA or RNA can be amplified by this method. More recently, real-time PCR has undergone significant development. By incorporating double-stranded DNA intercalating dye or fluorescently labeled oligonucleotides, PCR amplification and DNA or RNA detection occur simultaneously in the same reaction mixture. The change in fluorescence intensity is due to the presence of double-stranded DNA (double-stranded DNA binding dye), a loss of fluorescence quenching (TaqMan probe) or a gain of longer wavelength fluorescence through fluorescence resonance energy transfer (FRET) from a donor probe to an acceptor probe (hybridization probe). Recently, unlabeled oligonucleotides in the presence of saturating DNA dye have been used for single nucleotide polymorphism detection by high-resolution DNA melting analysis. The unlabeled oligonucleotides can act as a hybridization probe with the use of double-stranded DNA binding dye. The probe-amplicon duplexes melt at lower temperature than amplicon duplexes, and hence mutation scanning and genotyping can be observed in one melting curve analysis. During our previous study of hybridization probe assay and unlabeled probe assay, aberrant probe cleavage was sometimes observed which might affect the melting analysis. It was later found that the probes were hydrolyzed by the 5' nuclease activity of Taq DNA polymerase. The melting temperature of degraded probes was lower than that of the intact probe. The presence of degraded probes and intact probes in one mixture generated aberrant melting profiles that complicated assay interpretation. Locked nucleic acid residues, phosphorothioate internucleotide linkage, and mismatched bases to their complementary strand were reported to confer a relative degree of nuclease resistance. Here, we incorporated locked nucleic acid residues, phosphorothioate internucleotide linkage, mismatched nucleotides or a combination of them into 5' termini of probe sequences and studied their effects on 5' nuclease stability. After real-time PCR in LightCycler480, products were checked by agarose gel electrophoresis and studied by denaturating high performance liquid chromatography for probe alteration by comparing changes in peak profiles with reference chromatograms. The probes modified with phosphorothioate intemucleotide linkages, locked nucleic acids or phosphorothioate intemucleotide linkages combined with mismatched nucleotides showed increased enzymatic stability when the PCR were within 35 cycles. When PCR cycles went up to 45 cycles, all the modified probes exhibited some degree of degradation, and aberrant peaks were observed in peak profiles of melting curve analysis. We also found that different 3' end modifications (FAM labeled probe compared with probe with 3'phosphate) could affected the 5' nuclease activity of Taq DNA polymerase. Compared the hybridization affinity of modified probes with unmodified probes, the LNA-modified probes demonstrated the highest thermal stability towards their complementary DNA sequence while the phosphorothioate modification lowered the melting temperature about 1 oC per modification.
Rights: All rights reserved
Access: restricted access

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