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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.
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...
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...
RNA-seq03:21

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. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...

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Related Experiment Video

Updated: Jun 3, 2026

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
08:23

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Published on: September 25, 2018

Direct dideoxy DNA sequencing.

A Graham1, J Steven, D McKechnie

  • 1Biotechnology Division, Inveresk Research International Ltd., Musselburgh, Scotland.

Methods in Molecular Biology (Clifton, N.J.)
|March 23, 2011
PubMed
Summary
This summary is machine-generated.

Directly sequencing double-stranded DNA using chain terminators simplifies genetic analysis. This method avoids subcloning into M13 vectors, enabling efficient DNA sequencing from both ends of inserts.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Advances in DNA sequencing enable routine analysis of long DNA molecules.
  • Traditional methods involve subcloning into single-stranded M13 vectors, which is time-consuming.

Purpose of the Study:

  • To describe a method for direct sequencing of double-stranded DNA using chain terminators.
  • To eliminate the need for subcloning into M13 vectors.

Main Methods:

  • Denaturing a plasmid DNA template to separate strands.
  • Using a primer complementary to the plasmid vector adjacent to the cloning site.
  • Employing Klenow fragment of Escherichia coli DNA polymerase I or reverse transcriptase for primed synthesis.
  • Utilizing dideoxynucleoside triphosphates (ddNTPs) for chain termination at specific nucleotides.
  • Analyzing reaction products on DNA sequencing gels.

Main Results:

  • Successful direct sequencing of double-stranded DNA was achieved.
  • The method eliminates the requirement for subcloning into M13 vectors.
  • Sequencing from both ends of the DNA insert is possible by using primers for each strand.

Conclusions:

  • Direct sequencing of double-stranded DNA with chain terminators is a more efficient approach.
  • This technique simplifies the DNA sequencing workflow by removing intermediate steps.
  • The method allows for comprehensive sequence analysis of DNA inserts.