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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...
Next-generation Sequencing03:00

Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
Base Excision Repair01:54

Base Excision Repair

One of the common DNA damages is the chemical alteration of single bases by alkylation, oxidation, or deamination. The altered bases cause mispairing and strand breakage during replication. This type of damage causes minimal change to the DNA double helix structure and can be repaired by the base excision repair (BER) pathways. BER corrects damaged DNA sequences by removing the damaged base and restoring the original base sequence using the complementary strand as a template.
The first step of...
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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Homologous Recombination02:31

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
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Updated: Jul 7, 2026

Novel Sequence Discovery by Subtractive Genomics
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Published on: January 25, 2019

Constructing nested deletions for use in DNA sequencing.

B Slatko1, P Heinrich, B T Nixon

  • 1New England Biolabs, Beverly, Massachusetts, USA.

Current Protocols in Molecular Biology
|February 12, 2008
PubMed
Summary

Nested deletions enable dideoxy DNA sequencing by creating progressively longer DNA fragments. Exonuclease III and Bal 31 nuclease are two enzymatic methods for generating these essential sequencing tools.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Nested deletions are crucial for dideoxy DNA sequencing.
  • They create progressively longer DNA fragments for analysis.
  • This technique expands the sequencing range from a primer site.

Purpose of the Study:

  • To describe protocols for generating nested deletions.
  • To compare enzymatic methods for creating nested subclones.
  • To highlight applications in DNA sequencing.

Main Methods:

  • Generating nested deletions using Exonuclease III.
  • Generating nested deletions using Bal 31 nuclease.
  • Utilizing unique restriction sites in vectors for insert DNA.

Main Results:

  • Exonuclease III method allows recircularization of deletion products into functional plasmids.
  • Bal 31 nuclease method requires subcloning deletion fragments into a separate vector.
  • Both methods are effective for generating nested deletions for sequencing.

Conclusions:

  • Two distinct enzymatic protocols for generating nested deletions are presented.
  • The choice of method depends on downstream applications and vector requirements.
  • Nested deletions are a versatile tool for DNA sequencing and analysis.