in-solution hybridization capture
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Many approaches aimed at improving next-generation sequencing output for clinical purposes exist. However, sequencing gaps or misalignments for regions that are difficult to cover because of their low complexity or high homology still exist. Our aim was to improve the yield of sequencing data. A hybridization-based next-generation sequencing library was pooled with custom add-on amplicon-based libraries processed by the same commercial test and run in parallel and sequenced simultaneously. Formulas and steps for proper amplicon pooling (250 to 7000 bp) and final library merging are presented. The novel strategy was tested on selected archetypal situations: diagnostics of a gene with many pseudogenes, a genomic region surrounded by Alu repeats, simple one-time addition of an extra gene, and mosaicism detection. The sequence of all supplemented genomic regions was traced with reasonable coverage at the background of a hybridization captured library. The flexible add-on module expands the possibilities of routine diagnostics. The technical solution makes it possible to mix amplicons that differ significantly in size and process them in one tube simultaneously with samples of the hybridization-based panel. The proposed approach reduces turnaround time and increases diagnostic yield.
- MeSH
- genomika * MeSH
- genová knihovna MeSH
- hybridizace nukleových kyselin MeSH
- lidé MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.
... :rc Cells Evolved from a Symbiosis -a.e Hybrid Genomes Senomes Are Big -- :-mes Are Rich in Regulatory ... ... Can Be Determined Using X-Ray Diffraction 460 -- NMR Can Be Used to Determine Protein Structure in Solution ... ... DNA Library 469 -- Genomic and cDNA Libraries Have Different Advantages and -- Drawbacks 471 -- Hybridization ... ... Test Gene -- Function 499 -- Reporter Genes Reveal When and Where a Gene Is Expressed 501 In situ Hybridization ... ... Cytosolic pH emc Distribution of Transporters in Epithelial Cells :he Transcellular Transport of Solutes ...
Sixth edition xxxiv, 1430 stran v různém stránkování : ilustrace (převážně barevné) ; 29 cm
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- molekulární biologie, molekulární medicína
- NLK Publikační typ
- učebnice vysokých škol
... Genes Requires Ordered Binding and Function of Activators and Co-activators 308 -- The Yeast Two-Hybrid ... ... Can Grow Indefinitely in Culture 397 -- Some Cell Lines Undergo Differentiation in Culture 398 -- Hybrid ... ... Hybridomas Produce Abundant -- Monoclonal Antibodies 400 -- HAT Medium Is Commonly Used to Isolate Hybrid ... ... Proteins to the Membrane 427 -- Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions ... ... -- Individual Microtubules Exhibit Dynamic Instability 763 -- Localized Assembly and \"Search-and-Capture ...
6th ed. xxxvii, 1150 s. : il., tab. ; 29 cm
- MeSH
- biologie buňky MeSH
- molekulární biologie MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
- cytologie, klinická cytologie
... Problem 264 -- 8.5 DNA Arrays as an Alternative Sequencing Technique 265 -- 8.6 Sequencing by Hybridization ...
Computational molecular biology series
[1st ed.] xviii, 435 s. : il.
- MeSH
- algoritmy MeSH
- informatika MeSH
- Konspekt
- Lékařské vědy. Lékařství
- NLK Obory
- lékařská informatika
... Stark Ch., Vogl R., WiIhelmy 1 66 -- Innovations in Telemedicine: -- Richards В 67 -- A New Web Solution ... ... Votruba P.,Miksch S., Seyfang A., Kosara R 84 -- The Digital Electronic Guideline Library (DeGeL): A Hybrid ... ... R 112 -- Supporting Knowledge Capture and Learning: -- Zdráha! ... ... Architecture: -- Biobel В 169 -- Clinical Guidelines and Decision Support: -- Dudeck J 170 -- IHE - Solution-oriented ...
184 s. ; 22 cm
- MeSH
- lékařská informatika MeSH
- lékařství MeSH
- výpočetní biologie MeSH
- Publikační typ
- abstrakt z konference MeSH
- sborníky MeSH
- Konspekt
- Lékařské vědy. Lékařství
- NLK Obory
- lékařská informatika
... Cell Lines Are a Convenient Source of Homogeneous Cells 162 -- Cells Can Be Fused Together to Form Hybrid ... ... Be Cloned 188 xii List of Topics -- Cloned DNA Fragments Can Be Rapidly Sequenced Nucleic Acid Hybridization ... ... Reactions Pro\\ide a Sensitive Way of Detecting Specific Nucleotide Sequences -- In Situ Hybridization ... ... Membrane Na+-K+ Pump Is an ATPase -- The Na+-K+ ATPase Helps Control Cell Volume by Controlling the Solute ... ... Fixation Is Compartmentalized to Facilitate Growth at Low C02 Concentrations 516 -- Light Energy Captured ...
xxxix, 1146 s. : il., tab. ; 28 cm
- MeSH
- biologie buňky MeSH
- molekulární biologie MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- biologie
- cytologie, klinická cytologie
