Electrochemical Detection of SNP in Human Mitochondrial DNA Using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68081707%3A_____%2F18%3A00502202" target="_blank" >RIV/68081707:_____/18:00502202 - isvavai.cz</a>

Výsledek na webu

<a href="http://dx.doi.org/10.1002/elan.201800314" target="_blank" >http://dx.doi.org/10.1002/elan.201800314</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1002/elan.201800314" target="_blank" >10.1002/elan.201800314</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Electrochemical Detection of SNP in Human Mitochondrial DNA Using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes

Popis výsledku v původním jazyce

A novel method of SNP typing in human mitochondrial DNA utilizing enzymatic labeling and electrochemical detection at disposable pencil graphite electrodes is described. The procedure is based on amplification of DNA stretches by cyclic primer extension (PEx) of SNP-specific diagnostic primers in a mixture of biotinylated and natural nucleotides. The diagnostic primers are designed to recognize, by its 3'-terminal nucleotide, the SNP-site in target template. Under optimized conditions of the PEx reaction, efficient polymerase synthesis of biotin-labeled strands takes place only in the case of full complementarity between the diagnostic primer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNA strand, resulting in another level of signal amplification. After adsorption of biotinylated PEx products at the electrode surface, streptavidin-alkaline phosphatase conjugate was bound to the biotin tags, 1-naphthol was enzymatically produced and electrochemically detected. Several critical steps and parameters of the assay, including termination of 3'-OH ends of residual amplification primers, temperature for annealing of diagnostic primers, relative amount of biotinylated deoxynucleoside triphosphate in the PEx mixture and number of PEx cycles were optimized in this study to attain best SNP resolution, and reduction of time needed for the analysis.

Název v anglickém jazyce

Electrochemical Detection of SNP in Human Mitochondrial DNA Using Cyclic Primer Extension with Biotinylated Nucletides and Enzymatic Labeling at Disposable Pencil Graphite Electrodes

Popis výsledku anglicky

A novel method of SNP typing in human mitochondrial DNA utilizing enzymatic labeling and electrochemical detection at disposable pencil graphite electrodes is described. The procedure is based on amplification of DNA stretches by cyclic primer extension (PEx) of SNP-specific diagnostic primers in a mixture of biotinylated and natural nucleotides. The diagnostic primers are designed to recognize, by its 3'-terminal nucleotide, the SNP-site in target template. Under optimized conditions of the PEx reaction, efficient polymerase synthesis of biotin-labeled strands takes place only in the case of full complementarity between the diagnostic primer and the target SNP site. There is also benefit from introducing many biotin molecules per extended DNA strand, resulting in another level of signal amplification. After adsorption of biotinylated PEx products at the electrode surface, streptavidin-alkaline phosphatase conjugate was bound to the biotin tags, 1-naphthol was enzymatically produced and electrochemically detected. Several critical steps and parameters of the assay, including termination of 3'-OH ends of residual amplification primers, temperature for annealing of diagnostic primers, relative amount of biotinylated deoxynucleoside triphosphate in the PEx mixture and number of PEx cycles were optimized in this study to attain best SNP resolution, and reduction of time needed for the analysis.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10405 - Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis)

Projekt

<a href="/cs/project/GBP206%2F12%2FG151" target="_blank" >GBP206/12/G151: Centrum nových přístupů k bioanalýze a molekulární diagnostice</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2018

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Electroanalysis

ISSN

1040-0397

e-ISSN

—

Svazek periodika

30

Číslo periodika v rámci svazku

10

Stát vydavatele periodika

DE - Spolková republika Německo

Počet stran výsledku

9

Strana od-do

2321-2329

Kód UT WoS článku

000446660400016

EID výsledku v databázi Scopus

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