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Related Concept Videos

DNA Isolation01:24

DNA Isolation

DNA isolation protocols can be fast and straightforward or complex and time-consuming depending on the type and quality of DNA required for further processing. For example, plasmid DNA extraction is a bit more complicated than genomic DNA extraction because of the need for an appropriate lysis method to separate plasmid DNA from gDNA during isolation. However, for specific applications, such as long-range DNA sequencing that require a good yield of high- quality DNA samples, we need to follow...
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...
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.
Denatured DNA fragments must be transferred onto a carrier membrane from the gel to make it accessible to a probe - a small ssDNA fragment complementary to the target DNA...
Sanger Sequencing01:57

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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...
Modern Molecular Taxonomy01:29

Modern Molecular Taxonomy

Advancements in molecular biology have revolutionized the identification and characterization of bacteria, with multiple methods leveraging DNA sequencing for enhanced precision. As sequencing technologies improve and costs decline, these approaches are increasingly used in clinical, environmental, and evolutionary studies.Multilocus Sequence Typing (MLST) examines several housekeeping genes, essential chromosomal genes encoding cellular functions, to distinguish strains. Approximately...

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Related Experiment Video

Updated: Jun 18, 2026

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling
08:04

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

Published on: October 8, 2019

A simple, high-resolution method for establishing DNA binding affinity and sequence selectivity.

D L Boger1, B E Fink, S R Brunette

  • 1Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.

Journal of the American Chemical Society
|June 21, 2001
PubMed
Summary
This summary is machine-generated.

A new fluorescent intercalator displacement (FID) assay offers a simple, rapid, and cost-effective method for determining DNA binding affinity and sequence selectivity. This high-throughput technique profiles compound interactions with DNA, aiding drug discovery and development.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Drug Discovery

Background:

  • Assessing DNA binding affinity and sequence selectivity is crucial for understanding molecular interactions and developing therapeutic agents.
  • Existing methods like footprinting or affinity cleavage can be time-consuming and lack high resolution.
  • A need exists for a rapid, high-throughput, and cost-effective assay for DNA binding profiling.

Purpose of the Study:

  • To develop and validate a simple, nondestructive, and high-throughput method for establishing DNA binding affinity and sequence selectivity.
  • To demonstrate the utility of the fluorescent intercalator displacement (FID) assay for profiling various DNA-binding agents.
  • To compare quantitative titration methods for determining DNA binding constants.

Main Methods:

  • Development of a fluorescent intercalator displacement (FID) assay utilizing ethidium bromide or thiazole orange.
  • Employing libraries of hairpin deoxyoligonucleotides (512 or 136 sequences) in a 96-well format.
  • Assessing DNA binding affinity and sequence selectivity through fluorescence loss or change in intrinsic fluorescence.

Main Results:

  • The FID assay successfully generated rank-order binding profiles for compounds, defining sequence selectivity with high resolution.
  • The assay is cost-effective (ca. $100/assay), rapid (15 min readout), and automatable.
  • Profiles for distamycin A, netropsin, DAPI, Hoechst 33258, and berenil were generated, demonstrating the assay's applicability.

Conclusions:

  • The FID assay provides a powerful, high-resolution tool for characterizing DNA binding affinity and sequence selectivity.
  • This method complements existing techniques and offers advantages in speed, cost, and throughput.
  • The assay is valuable for drug discovery, enabling efficient screening and profiling of DNA-interacting compounds.