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The Sanger sequencing method creates large numbers of sequences of all possible lengths, ending with a specific nucleotide, by terminating with a tagged (fluorescent) nucleotide at the end.

But if you already have fluorescent nucleotides of a specific base, why not just do regular PCR with them, create a huge number of full length copies of the original sequence, and then simply see the locations where the nucleotide fluoresces to determine all the locations of that base. Why do we need a large number of copies ending at each possible location, as with the Sanger method?

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    $\begingroup$ It is unclear whether you are asking a) how the Sanger method works, or b) why it is used (or why it was invented) when Next Gen methods are better. However my guess is that you are student who has encountered DNA sequencing methods without any historical or practical context, and has got bogged down in the preparation of the samples rather than the methods for actually determining the sequences. Read Wikipedia on Sanger and more modern Sequencing methods. (The Sanger method predates PCR.) $\endgroup$ – David Sep 12 at 9:46
  • $\begingroup$ > why not just do regular PCR with them, create a huge number of full > length copies of the original sequence, and then simply see the > locations where the nucleotide fluoresces to determine all the > locations of that base. How? What instrument do you propose to use to see the sequence of fluorescent tags? $\endgroup$ – swbarnes2 Sep 12 at 16:04
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If you mark the full length strand of the DNA with the fluorescent labels, you will get a lot of signals from the same nucleotide without the possibility to discriminate where the actual nucleotide is located on the strand.

Sanger sequencing doesn't end with the preparation of the terminated and labelled DNA strands, the following step is crucial in discriminating where the labelled base actually is. After labelling, the sample is run through high resolution capillary gel electrophoresis to sort them by size. The smallest fragment comes out first, then the one with one base more and so on. The detector which then identifies which fragment is coming through is located at the end of the capillary.

This works like shown in the schematic image (from here):

enter image description here

If you would mark a complete strand, no dicrimination by size is possible (ideally, all DNA would run on the same height of a gel) and all flourescent signals for all positions would come on the same spot. Not very helpful.

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    $\begingroup$ Additionally, the original method of Sanger didn’t use labelled ddNTPs at all. The sequence was determined solely on the basis of fragment size and knowledge of which ddNTP terminated it. $\endgroup$ – canadianer Sep 12 at 22:05
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    $\begingroup$ And the Sanger method didn't use capilliary gel electrophoresis for many years after its inception. The principle is merely separation, which in a single small laboratory is proably still done by slab gel electrophoresis. $\endgroup$ – David Sep 23 at 12:14
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Foreword

I originally suggested closing this question as I think it confuses methodological details with strategy. There is an accepted answer that clarifies things with a clear diagram of the Sanger method. In the circumstances I decided to add a different sort of answer — one that emphasises the principles involved in the different approaches, rather than practical details which are not intimately associated with logic of the different approaches.

General Strategy of DNA sequencing

Irrespective of the method, the determination of DNA (or any linear) sequence requires two pieces of conceptual information:

  1. Knowledge of a particular position in the sequence.
  2. The identity of the base at that position. This is illustrated in the graphic. In addition, there is a practical requirement:
  3. Means to uniquely detect this.

Sequencing Strategy

Let us compare these in three different sequencing approaches.

A. Chemical fragmentation (Maxam & Gilbert)

Here the DNA to be sequenced labelled at one end is treated with different chemicals that cleave at different bases in separate reactions.

  1. The position is indicated by the length of the fragment.
  2. The base is known from the chemical used.
  3. Detection is performed by separating the different fragments by length and using their radioactivity to visualize them (by autoradiography).

B. Chain termination during synthesis (Sanger)

This method uses enzyme-catalysed copying of the DNA to be sequenced, with termination of the copying produced by the four di-deoxy analogues of the deoxynucleotide triphosphates (dNTPs) in separate reactions.

  1. The position is indicated by the length of the fragment (as in A).
  2. The base is known from the particular di-deoxy analogue used to terminate the reaction.
  3. Detection is performed by separating the products of synthesis by length and using the radioactivity or fluorescence of the dNTPs used in synthesis to visualize them.

(Note to the poster: obviously you need fragments of all different lengths if you are going to identify the base at each the position in the chain.)

C. Phased synthesis (‘Next Generation Sequencing’)

This type of method also uses enzyme-catalysed copying of the DNA to be sequenced. However, there is no termination of the chains, as in B, but the cycles of addition are followed by various techniques.

  1. The position of a base in a sequence is indicated by the cycle of addition at which the insertion of the base is detected.
  2. The base is known from which of the different dNTPs allow the extension reaction to occur.
  3. Detection methods vary but include detecting the release of pyrophosphate by coupling to a light-emitting reaction (pyrosequencing) and detecting hydrogen ions released during the polymerization using a semiconductor chip (ion torrent).

Coda: Principle and Practice

In stressing the general principles of DNA sequencing the methodology mentioned has been limited to that required for detection — there was no need to even mention PCR. Of course, different methodologies for preparing multiple samples for sequencing have hugely influenced the speed and cost of DNA sequencing. However the student should understand the principles before getting bogged down in the details, which can be found e.g. in this Wikipedia article on sequencing and on the website of ATDBIO (albeit a not disinterested company).

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