Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage.
Clin Chem
; 69(4): 363-373, 2023 04 03.
Article
en En
| MEDLINE
| ID: mdl-36807661
ABSTRACT
BACKGROUND:
Isothermal amplification is considered to be one of the most promising tools for point-of-care testing molecular diagnosis. However, its clinical application is severely hindered by nonspecific amplification. Thus, it is important to investigate the exact mechanism of nonspecific amplification and develop a high-specific isothermal amplification assay.METHODS:
Four sets of primer pairs were incubated with Bst DNA polymerase to produce nonspecific amplification. Gel electrophoresis, DNA sequencing, and sequence function analysis were used to investigate the mechanism of nonspecific product generation, which was discovered to be nonspecific tailing and replication slippage mediated tandem repeats generation (NT&RS). Using this knowledge, a novel isothermal amplification technology, bridging primer assisted slippage isothermal amplification (BASIS), was developed.RESULTS:
During NT&RS, the Bst DNA polymerase triggers nonspecific tailing on the 3'-ends of DNAs, thereby producing sticky-end DNAs over time. The hybridization and extension between these sticky DNAs generate repetitive DNAs, which can trigger self-extension via replication slippage, thereby leading to nonspecific tandem repeats (TRs) generation and nonspecific amplification. Based on the NT&RS, we developed the BASIS assay. The BASIS is carried out by using a well-designed bridging primer, which can form hybrids with primer-based amplicons, thereby generating specific repetitive DNA and triggering specific amplification. The BASIS can detect 10 copies of target DNA, resist interfering DNA disruption, and provide genotyping ability, thereby offering 100% accuracy for type 16 human papillomavirus detection.CONCLUSION:
We discovered the mechanism for Bst-mediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity.Palabras clave
Texto completo:
1
Banco de datos:
MEDLINE
Asunto principal:
ADN
/
Técnicas de Amplificación de Ácido Nucleico
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Clin Chem
Asunto de la revista:
QUIMICA CLINICA
Año:
2023
Tipo del documento:
Article