Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture.

ABSTRACT: Arrays of short, singly-labeled ssDNA oligonucleotides enable in situ hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way.

SUBMITTER: Gaspar I

PROVIDER: S-EPMC8203885 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Terminal Deoxynucleotidyl Transferase Mediated Production of Labeled Probes for Single-molecule FISH or RNA Capture.

Gaspar Imre I Wippich Frank F Ephrussi Anne A

Bio-protocol 20180305 5

Arrays of short, singly-labeled ssDNA oligonucleotides enable <i>in situ</i> hybridization with single molecule sensitivity and efficient transcript specific RNA capture. Here, we describe a simple, enzymatic protocol that can be carried out using basic laboratory equipment to convert arrays of PCR oligos into smFISH and RAP probesets in a quantitative, cost-efficient and flexible way. ...[more]

PMID: 34179277

Similar Datasets

Enzymatic production of single-molecule FISH and RNA capture probes.

Project description:Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed.

| S-EPMC5602115 | biostudies-literature

Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides.

Project description:Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

| S-EPMC7826267 | biostudies-literature

Evolving a terminal deoxynucleotidyl transferase for commercial enzymatic DNA synthesis

Project description: Not available

| S-EPMC11840559 | biostudies-literature

Terminal deoxynucleotidyl transferase: the story of a misguided DNA polymerase.

Project description:Nearly every DNA polymerase characterized to date exclusively catalyzes the incorporation of mononucleotides into a growing primer using a DNA or RNA template as a guide to direct each incorporation event. There is, however, one unique DNA polymerase designated terminal deoxynucleotidyl transferase that performs DNA synthesis using only single-stranded DNA as the nucleic acid substrate. In this chapter, we review the biological role of this enigmatic DNA polymerase and the biochemical mechanism for its ability to perform DNA synthesis in the absence of a templating strand. We compare and contrast the molecular events for template-independent DNA synthesis catalyzed by terminal deoxynucleotidyl transferase with other well-characterized DNA polymerases that perform template-dependent synthesis. This includes a quantitative inspection of how terminal deoxynucleotidyl transferase binds DNA and dNTP substrates, the possible involvement of a conformational change that precedes phosphoryl transfer, and kinetic steps that are associated with the release of products. These enzymatic steps are discussed within the context of the available structures of terminal deoxynucleotidyl transferase in the presence of DNA or nucleotide substrate. In addition, we discuss the ability of proteins involved in replication and recombination to regulate the activity of the terminal deoxynucleotidyl transferase. Finally, the biomedical role of this specialized DNA polymerase is discussed focusing on its involvement in cancer development and its use in biomedical applications such as labeling DNA for detecting apoptosis.

| S-EPMC2846215 | biostudies-literature

Terminal deoxynucleotidyl transferase and CD84 identify human multi-potent lymphoid progenitors.

Project description:Lymphoid specification in human hematopoietic progenitors is not fully understood. To better associate lymphoid identity with protein-level cell features, we conduct a highly multiplexed single-cell proteomic screen on human bone marrow progenitors. This screen identifies terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase intrinsic to VDJ recombination, broadly expressed within CD34+ progenitors prior to B/T cell emergence. While these TdT+ cells coincide with granulocyte-monocyte progenitor (GMP) immunophenotype, their accessible chromatin regions show enrichment for lymphoid-associated transcription factor (TF) motifs. TdT expression on GMPs is inversely related to the SLAM family member CD84. Prospective isolation of CD84lo GMPs demonstrates robust lymphoid potentials ex vivo, while still retaining significant myeloid differentiation capacity, akin to LMPPs. This multi-omic study identifies human bone marrow lymphoid-primed progenitors, further defining the lympho-myeloid axis in human hematopoiesis.

| S-EPMC11246490 | biostudies-literature

Recording Binding Information Directly into DNA-Encoded Libraries Using Terminal Deoxynucleotidyl Transferase.

Project description:Terminal deoxynucleotidyl transferase (TdT) is an unusual DNA polymerase that adds untemplated dNTPs to 3'-ends of DNA. If a target protein is expressed as a TdT fusion and incubated with a DNA-encoded library (DEL) in the presence of dATP, the binders of the target induce proximity between TdT and the DNA, promoting the synthesis of a poly-adenine (polyA) tail. The polyA tail length is proportional to the binding affinity, effectively serving as a stable molecular record of binding events. The polyA tail is also a convenient handle to enrich binders with magnetic poly(dT)25 beads before sequencing. In a benchmarking system, we show that ligands spanning nanomolar to double-digit micromolar binding can be cleanly identified by TdT extension, whereas only the tightest binding ligands are identified by classical affinity selection. The method is simple to implement and can function on any DEL that bears a free 3'-end.

| S-EPMC10540198 | biostudies-literature

Enzymatic synthesis and modification of high molecular weight DNA using terminal deoxynucleotidyl transferase.

Project description:The recognition that nucleic acids can be used as polymeric materials led to the blossoming of the field of DNA nanotechnology, with a broad range of applications in biotechnology, biosensors, diagnostics, and drug delivery. These applications require efficient methods to synthesize and chemically modify high molecular weight DNA. Here, we discuss terminal deoxynucleotidyl transferase (TdT)-catalyzed enzymatic polymerization (TcEP) as an alternative to conventional enzymatic and solid-phase DNA synthesis. We describe biochemical requirements for TcEP and provide step-by-step protocols to carry out TcEP in solution and from surfaces.

| S-EPMC7241426 | biostudies-literature

Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3' Terminal Structures for Enzymatic De Novo DNA Synthesis.

Project description:Enzymatic oligonucleotide synthesis methods based on the template-independent polymerase terminal deoxynucleotidyl transferase (TdT) promise to enable the de novo synthesis of long oligonucleotides under mild, aqueous conditions. Intermediates with a 3' terminal structure (hairpins) will inevitably arise during synthesis, but TdT has poor activity on these structured substrates, limiting its usefulness for oligonucleotide synthesis. Here, we described two parallel efforts to improve the activity of TdT on hairpins: (1) optimization of the concentrations of the divalent cation cofactors and (2) engineering TdT for enhanced thermostability, enabling reactions at elevated temperatures. By combining both of these improvements, we obtained a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin.

| S-EPMC7016565 | biostudies-literature

Role of human Pso4 in mammalian DNA repair and association with terminal deoxynucleotidyl transferase.

Project description:Terminal deoxynucleotidyl transferase (TdT; EC 2.7.7.31) adds nucleotides to DNA ends generated during V(D)J recombination that are subsequently processed by proteins involved in general double-strand break (DSB) repair pathways. We report an association between TdT and a 55-kDa protein in lymphoid cells. This protein, identified as hPso4, is a homolog of the protein encoded by the PS04/PRP19 gene in Saccharomyces cerevisiae that has pleiotropic functions in DNA recombination and error-prone repair. Purified hPso4 binds double-stranded DNA in a sequence-nonspecific manner but does not bind single-stranded DNA. hPso4 protein is induced 15- to 30-fold in cells by gamma radiation and chemical mutagens but not by UV treatment. Loss of hPso4 expression induced by siRNA results in accumulation of DSBs, apoptosis, and decreased cell survival after DNA damage. We conclude that hPso4 plays a major and previously undefined role in mammalian DNA DSB repair.

| S-EPMC196874 | biostudies-literature

Terminal deoxynucleotidyl transferase is required for an optimal response to the polysaccharide α-1,3 dextran.

Project description:An understanding of Ab responses to polysaccharides associated with pathogenic microorganisms is of importance for improving vaccine design, especially in neonates that respond poorly to these types of Ags. In this study, we have investigated the role of the lymphoid-specific enzyme TdT in generating B cell clones responsive to alpha-1,3 dextran (DEX). TdT is a DNA polymerase that plays a major role in generating diversity of lymphocyte AgRs during V(D)J recombination. In this study, we show that the DEX-specific Ab response is lower, and the dominant DEX-specific J558 idiotype (Id) is not detected in TdT(-/-) mice when compared with wild-type (WT) BALB/c mice. Nucleotide sequencing of H chain CDR3s of DEX-specific plasmablasts, sorted postimmunization, showed that TdT(-/-) mice generate a lower frequency of the predominant adult molecularly determined clone J558. Complementation of TdT expression in TdT(-/-) mice by early forced expression of the short splice variant of TdT-restored WT proportions of J558 Id+ clones and also abrogated the development of the minor M104E Id+ clones. J558 Id V(D)J rearrangements are detected as early as 7 d after birth in IgM-negative B cell precursors in the liver and spleen of WT and TdT-transgenic mice but not in TdT(-/-) mice. These data show that TdT is essential for the generation of the predominant higher-affinity DEX-responsive J558 clone.

| S-EPMC4710363 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data