The previously reported approach of orthogonal multipotential redox coding of all four DNA bases allowed only analysis of the relative nucleotide composition of short DNA stretches. Here, we present two methods for normalization of the electrochemical readout to facilitate the determination of the total nucleotide composition. The first method is based on the presence or absence of an internal standard of 7-deaza-2'-deoxyguanosine in a DNA primer. The exact composition of the DNA was elucidated upon two parallel analyses and the subtraction of the electrochemical signal intensities. The second approach took advantage of a 5'-viologen modified primer, with this fifth orthogonal redox label acting as a reference for signal normalization, thus allowing accurate electrochemical sequence analysis in a single read. Both approaches were tested using various sequences, and the voltammetric signals obtained were normalized using either the internal standard or the reference label and demonstrated to be in perfect agreement with the actual nucleotide composition, highlighting the potential for targeted DNA sequence analysis.

• MeSH
• DNA primery MeSH
• DNA * chemie   MeSH
• nukleotidy * chemie   MeSH
• oxidace-redukce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• DNA * MeSH
• nukleotidy * MeSH

Osteoporosis is a multifactorial disease influenced by genetic and environmental factors, which contributes to an increased risk of bone fracture, but early diagnosis of this disease cannot be achieved using current techniques. We describe a generic platform for the targeted electrochemical genotyping of SNPs identified by genome-wide association studies to be associated with a genetic predisposition to osteoporosis. The platform exploits isothermal solid-phase primer elongation with ferrocene-labeled nucleoside triphosphates. Thiolated reverse primers designed for each SNP were immobilized on individual gold electrodes of an array. These primers are designed to hybridize to the SNP site at their 3'OH terminal, and primer elongation occurs only where there is 100% complementarity, facilitating the identification and heterozygosity of each SNP under interrogation. The platform was applied to real blood samples, which were thermally lysed and directly used without the need for DNA extraction or purification. The results were validated using Taqman SNP genotyping assays and Sanger sequencing. The assay is complete in just 15 min with a total cost of 0.3€ per electrode. The platform is completely generic and has immense potential for deployment at the point of need in an automated device for targeted SNP genotyping with the only required end-user intervention being sample addition.

• Publikační typ
• časopisecké články MeSH

We designed and synthesized a series of 2'-deoxyribonucleoside triphosphates (dNTPs) bearing various lipid moieties. Fatty acid- and cholesterol-modified dNTPs proved to be substrates for KOD XL DNA polymerase in primer extension reactions. They were also mutually compatible for simultaneous multiple incorporations into the DNA strand. The methodology of enzymatic synthesis opened a pathway to diverse structurally unique lipid-ON probes containing one or more lipid units. We studied interactions of such probes with the plasma membranes of live cells. Employing a rational design, we found a series of lipid-ONs with enhanced membrane anchoring efficiency. The in-membrane stability of multiply modified ONs was superior to that of commonly studied ON analogues, in which a single cholesterol molecule is typically tethered to the thread end. Notably, some of the probes were detected at the cell surface even after 24 h upon removal of the probe solution. Such an effect was general to several studied cell lines.

• Publikační typ
• časopisecké články MeSH

Here, we report the electrochemical detection of single-point mutations using solid-phase isothermal primer elongation with redox-labeled oligonucleotides. A single-base mutation associated with resistance to rifampicin, an antibiotic commonly used for the treatment of Mycobacterium tuberculosis, was used as a model system to demonstrate a proof-of-concept of the approach. Four 5'-thiolated primers, designed to be complementary with the same fragment of the target sequence and differing only in the last base, addressing the polymorphic site, were self-assembled via chemisorption on individual gold electrodes of an array. Following hybridization with single-stranded DNA, Klenow (exo-) DNA polymerase-mediated primer extension with ferrocene-labeled 2'-deoxyribonucleoside triphosphates (dNFcTPs) was only observed to proceed at the electrode where there was full complementarity between the surface-tethered probe and the target DNA being interrogated. We tested all four ferrocenylethynyl-linked dNTPs and optimized the ratio of labeled/natural nucleotides to achieve maximum sensitivity. Following a 20 min hybridization step, Klenow (exo-) DNA polymerase-mediated primer elongation at 37 °C for 5 min was optimal for the enzymatic incorporation of a ferrocene-labeled nucleotide, achieving unequivocal electrochemical detection of a single-point mutation in 14 samples of genomic DNA extracted from Mycobacterium tuberculosis strains. The approach is rapid, cost-effective, facile, and can be extended to multiplexed electrochemical single-point mutation genotyping.

• Klíčová slova
• Klenow (exo-) DNA polymerase, ferrocene-labeled nucleotides, nucleoside triphosphates, single-nucleotide polymorphism (SNP), single-point mutation, solid-phase primer elongation,
• MeSH
• jednonukleotidový polymorfismus MeSH
• metaloceny MeSH
• Mycobacterium tuberculosis * genetika   MeSH
• oxidace-redukce MeSH
• rifampin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• metaloceny MeSH
• rifampin MeSH

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

• MeSH
• adenin chemie   metabolismus   MeSH
• aptamery nukleotidové chemická syntéza   genetika   MeSH
• cytosin chemie   metabolismus   MeSH
• deoxyribonukleosidy chemie   genetika   metabolismus   MeSH
• dinukleosidfosfáty chemie   genetika   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy genetika   metabolismus   MeSH
• DNA chemie   genetika   metabolismus   MeSH
• guanin chemie   metabolismus   MeSH
• hydrofobní a hydrofilní interakce MeSH
• párování bází MeSH
• polymerázová řetězová reakce MeSH
• polymery chemická syntéza   metabolismus   MeSH
• replikace DNA * MeSH
• sekvence nukleotidů MeSH
• uracil chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenin MeSH
• aptamery nukleotidové MeSH
• cytosin MeSH
• deoxyribonukleosidy MeSH
• dinukleosidfosfáty MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• guanin MeSH
• polymery MeSH
• uracil MeSH