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Related Concept Videos

In-situ Hybridization02:31

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
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A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
Southern Blot02:57

Southern Blot

Agarose gel electrophoresis is very useful in separating DNA fragments by size. Running a DNA ladder containing fragments of the known length alongside the sample helps determine the approximate length of the sample DNA fragments. However, additional steps are needed to verify the sequence identity of the sample DNA fragments.
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Labeling DNA Probes03:31

Labeling DNA Probes

DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Histone Modification02:32

Histone Modification

The histone proteins have a flexible N-terminal tail extending out from the nucleosome. These histone tails are often subjected to post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Particular combinations of these modifications form “histone codes” that influence the chromatin folding and tissue-specific gene expression.
Acetylation
The enzyme histone acetyltransferase adds acetyl group to the histones. Another enzyme, histone deacetylase,...
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...

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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues
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Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

Published on: November 22, 2014

Hybrid activation methods for elucidating nucleic acid modifications.

Suncerae I Smith1, Jennifer S Brodbelt

  • 1Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, United States.

Analytical Chemistry
|December 15, 2010
PubMed
Summary
This summary is machine-generated.

Hybrid tandem mass spectrometry (MS/MS) methods using electron transfer (ET) combined with infrared multiphoton dissociation (IRMPD) or ultraviolet photodissociation (UVPD) effectively characterize modified oligonucleotides. These techniques provide detailed fragmentation for site-specific identification of modifications in DNA and RNA.

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Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes
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Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology

Background:

  • Oligonucleotides and oligoribonucleotides are crucial in biological processes and therapeutics.
  • Characterizing modified nucleic acids, such as those with platinated, phosphorothioated, or 2'-O-methylated sites, is essential for understanding their function and developing new treatments.
  • Existing mass spectrometry (MS) techniques may not provide sufficient detail for comprehensive analysis of modified oligonucleotides.

Purpose of the Study:

  • To evaluate hybrid tandem mass spectrometry (MS/MS) techniques for characterizing modified oligonucleotides and oligoribonucleotides.
  • To assess the fragmentation patterns and site-specific identification capabilities of electron transfer combined with different dissociation methods (IRMPD, UVPD).

Main Methods:

  • Implementation and evaluation of hybrid MS/MS techniques combining electron transfer (ET) with collision activated dissociation (CAD), infrared multiphoton dissociation (IRMPD), or ultraviolet photodissociation (UVPD).
  • Analysis of native single-stranded oligonucleotides and oligoribonucleotides containing various chemical modifications.
  • Characterization of fragmentation patterns, including the types of ions produced (e.g., w, a, z, d, c, y, a-B) and their ability to retain modification information.

Main Results:

  • ET-IRMPD and ET-UVPD yielded rich fragmentation spectra for both DNA and RNA, producing characteristic ions.
  • Many product ions retained modification information, enabling site-specific identification of modifications.
  • ET-IRMPD resulted in extensive secondary dissociation and a broader distribution of sequence ions.
  • ET-UVPD generated higher energy fragmentation, leading to the most diverse MS/MS spectra.
  • The hybrid MS/MS techniques achieved specific and extensive backbone cleavages, allowing elucidation of modification sites in multiply modified oligonucleotides.

Conclusions:

  • Hybrid ET-IRMPD and ET-UVPD MS/MS are powerful tools for the detailed characterization of modified oligonucleotides and oligoribonucleotides.
  • These methods facilitate site-specific identification of various modifications on nucleic acid strands.
  • The diverse fragmentation patterns obtained enhance the ability to elucidate the structure and modification status of complex nucleic acid molecules.