Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage.
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 amplifi...
| Publicado en: | Clinical Chemistry Vol. 69; no. 4; pp. 363 - 374 |
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| Autores principales: | , , , , , , , , , , |
| Formato: | Journal Article |
| Publicado: |
Oxford University Press / USA
Apr2023
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| Acceso en línea: | Ver este registro en EBSCOhost |
| fields | @attributes: recordID: 1 pdfLink: plink: https://search.ebscohost.com/login.aspx?direct=true&db=ccm&AN=162983118&site=ehost-live header: @attributes: shortDbName: ccm uiTerm: 162983118 longDbName: CINAHL Complete uiTag: AN controlInfo: bkinfo: dissinfo: jinfo: jid: 00099147 10CS jtl: Clinical Chemistry issn: 00099147 maglogo: N pubinfo: dt: Apr2023 vid: 69 iid: 4 pid: 622 pub: Oxford University Press / USA artinfo: ui: 162983118 10.1093/clinchem/hvac199 162983118 ppf: 363 ppct: 11 formats: fmt: @attributes: type: P tig: atl: Tandem Repeat Generation and Novel Isothermal Amplification Based on Nonspecific Tailing and Replication Slippage. aug: au: Guangcheng Luo Hongfei He Dongsheng Wang Shanshan Liu Shisu Tian Miaomiao Chen Qiang Wang Changsong Zhao Zhengwei Leng Lingmi Hou Xiaolan Guo affil: Department of Clinical Laboratory, Affiliated Hospital of North Sichuan Medical College, Nanchong, People's Republic of China sug: ab: 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 Bstmediated nonspecific TRs generation and developed a novel isothermal amplification assay (BASIS), which can detect nucleic acids with high sensitivity and specificity. pubtype: Academic Journal doctype: Journal Article ougenre: Article language: English refInfo: holdings: @attributes: islocal: N |
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