Designed phosphate-methylated oligonucleotides as PCR primers for SNP discrimination.
Anal Bioanal Chem
; 411(17): 3871-3880, 2019 Jul.
Article
en En
| MEDLINE
| ID: mdl-31209551
ABSTRACT
Polymerase chain reaction (PCR) is a powerful technique for the detection and quantification of nucleic acids and has enormous applications to research in molecular biology. Certain inherited diseases, caused by single nucleotide mutations, however, are difficult to identify by PCR, using DNA primers and probes, in a situation where a false diagnosis may lead to incorrect or delayed treatment. With the aim of enhancing the specificity of PCR, we used novel chemically synthesized oligonucleotides containing site-specific methyl phosphotriester (MPTE) inter-nucleoside linkage(s) as primers and probes. The methyl phosphotriester linkages carry no charge, so the reduction in the electrostatic repulsion of an MPTE-DNA/DNA duplex shows stronger hybridization affinity compared to a DNA/DNA duplex. However, the electrosteric effects introduced by the methyl group may result in instability of the double-stranded DNA (dsDNA) formed. With the use of specific MPTE modification sites and optimization of the number of MPTE modifications, greater delta melting temperature (ΔTm) may be obtained, in concert with adjustment of PCR operating conditions, especially with respect to the annealing temperature, to achieve more discriminatory results between the target template and the perfectly matched primer and the mismatched primer. In single nucleotide polymorphism (SNP) genotyping, the results demonstrated that MPTE-modified probes can improve specificity. In summary, MPTE-modified oligonucleotides are a promising DNA analog applied to PCR primers and probes to enhance the specificity and to provide more precise detection results for various applications, such as for genetic diagnosis. In summary, two common DNA polymerases we tested could successfully recognize the MPTE-modified primers and probes. Under the optimal operating conditions, MPTE modification has the ability to improve the discrimination of single nucleotide polymorphism by increasing the ΔTm of the perfect match and mismatch sequences and to provide more precise detection results for various applications, such as genetic diagnosis.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Fosfatos
/
Cartilla de ADN
/
Polimorfismo de Nucleótido Simple
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Reacción en Cadena en Tiempo Real de la Polimerasa
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Idioma:
En
Revista:
Anal Bioanal Chem
Año:
2019
Tipo del documento:
Article
País de afiliación:
Taiwán