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Related Concept Videos

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.
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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...
Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
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Next-generation Sequencing

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Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.

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Related Experiment Video

Updated: Jul 2, 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

Single-nucleotide sequence discrimination in situ using padlock probes.

M Nilsson1, U Landegren, D O Antson

  • 1Uppsala University, Uppsala, Sweden.

Current Protocols in Cytometry
|September 5, 2008
PubMed
Summary
This summary is machine-generated.

Short oligonucleotide probes improve DNA sequence discrimination in situ. These allele-specific probes offer enhanced sensitivity to mismatches, overcoming limitations of standard fluorescence in situ hybridization (FISH) methods.

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Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations
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Published on: March 29, 2017

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Standard fluorescence in situ hybridization (FISH) using long cloned probes struggles to differentiate similar DNA sequences due to low sensitivity to single base mismatches.
  • Distinguishing closely related DNA sequences in situ is crucial for various genetic analyses.

Purpose of the Study:

  • To present revised and expanded protocols for discriminating between closely similar DNA sequences in situ.
  • To introduce the use of short allele-specific oligonucleotide probes for improved sequence differentiation.

Main Methods:

  • Utilizing short allele-specific oligonucleotide probes that exhibit higher sensitivity to single base mismatches compared to traditional long probes.
  • Implementing revised protocols for in situ hybridization, including expanded discussions on probe synthesis.
  • Incorporating an alternate protocol for enzymatic probe ligation at low probe concentrations.
  • Describing a new support protocol for enzymatic probe synthesis.

Main Results:

  • Allele-specific oligonucleotide probes demonstrate superior ability to distinguish between closely similar DNA sequences in situ.
  • The revised protocols enhance the sensitivity and accuracy of in situ hybridization for sequence discrimination.
  • Optimized probe synthesis and ligation methods facilitate the application of these techniques.

Conclusions:

  • Short allele-specific oligonucleotide probes represent a significant advancement over standard FISH for precise DNA sequence analysis in situ.
  • The presented protocols provide researchers with improved tools for genetic analysis requiring high-resolution sequence discrimination.
  • These methods are valuable for applications in diagnostics, research, and understanding genetic variations.