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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...
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...
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...

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DNAzyme 10-23 - Based Nanomachines for Nucleic Acid Recognition
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DNA sequence determination using dideoxy analogs.

M J Owen1

  • 1ICRF Tumor Immunology Unit, Department of Zoology, University College London, London, England.

Methods in Molecular Biology (Clifton, N.J.)
|March 5, 2011
PubMed
Summary
This summary is machine-generated.

Sanger sequencing, a dideoxy chain termination method, accurately determines DNA sequences by halting DNA synthesis with dideoxynucleotides. This enables rapid sequencing of thousands of DNA bases daily.

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Pyrosequencing for Microbial Identification and Characterization
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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Dideoxy chain termination DNA sequencing, developed by Sanger and colleagues, is a highly accurate method for DNA sequence determination.
  • The technique allows for the sequencing of thousands of DNA bases per day.

Purpose of the Study:

  • To describe the principle and methodology of dideoxy chain termination DNA sequencing.
  • To explain how this method facilitates rapid and accurate DNA sequence acquisition.

Main Methods:

  • Utilizes a single-stranded DNA template and a complementary primer.
  • Employs DNA polymerase (Klenow fragment) and nucleoside triphosphates (dNTPs), with one labeled.
  • Incorporates dideoxynucleoside triphosphates (ddNTPs) to induce chain termination at specific nucleotides.
  • Separates terminated DNA chains by length using polyacrylamide gel electrophoresis.
  • Deduces the DNA sequence from the pattern of terminated chains across four reaction tracks.

Main Results:

  • Achieves specific chain termination at each of the four nucleotide types due to the absence of a 3' hydroxyl group in ddNTPs.
  • Generates DNA fragments of varying lengths that can be separated and analyzed.
  • Enables the determination of DNA sequence by reading the order of bands on an autoradiograph.

Conclusions:

  • Dideoxy chain termination sequencing is a robust and efficient method for obtaining extensive DNA sequence data.
  • The technique's accuracy and speed make it invaluable for genetic research and analysis.