Carboxymethylcellulose enhances the production of single-stranded DNA aptamers generated by asymmetric PCR

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F68378050%3A_____%2F20%3A00531045" target="_blank" >RIV/68378050:_____/20:00531045 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/abs/pii/S0003269719309169" target="_blank" >https://www.sciencedirect.com/science/article/abs/pii/S0003269719309169</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.ab.2019.113502" target="_blank" >10.1016/j.ab.2019.113502</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Carboxymethylcellulose enhances the production of single-stranded DNA aptamers generated by asymmetric PCR

Popis výsledku v původním jazyce

Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulosederived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.

Název v anglickém jazyce

Carboxymethylcellulose enhances the production of single-stranded DNA aptamers generated by asymmetric PCR

Popis výsledku anglicky

Nucleic acid aptamers are single-stranded (ss)DNA or RNA oligonucleotides that can take various conformations and bind specifically and with high affinity to selected targets. While the introduction of SELEX (systematic evolution of ligands by exponential enrichment) revolutionized the production of the aptamers, this procedure is impeded by the formation of undesirable by-products reflecting hybridization among complementary oligonucleotides in the ssDNA libraries during asymmetric PCR. To reduce nonspecific amplification we tested cellulosederived compounds and found that sodium carboxymethylcellulose (CMC) at a concentration 0.05%-0.2% efficiently suppressed production of undesirable large DNA amplicons during asymmetric PCR in the course of SELEX. Formation of the PCR by-products was reduced by CMCs of low and medium viscosity more than by CMCs of high viscosity, and all of them bound to DNA oligonucleotides as determined by electrophoresis in agarose gels. In contrast to CMC, methylcellulose did not reduce the formation of the PCR by-products and did not bind to DNA. DNA aptamers selected in the presence of CMC could be used directly in enzyme-linked immunosorbent-like assay. The combined data suggest that CMC binds weekly to DNA oligonucleotides through hydroxyl groups and in this way inhibits low-affinity DNA-DNA hybridization and enhances the production of specific amplicons in asymmetric PCR.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2020

Kód důvěrnosti údajů

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

Název periodika

Analytical Biochemistry

ISSN

0003-2697

e-ISSN

—

Svazek periodika

589

Číslo periodika v rámci svazku

589

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

6

Strana od-do

113502

Kód UT WoS článku

000518179100009

EID výsledku v databázi Scopus

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