Sangers method

Introduction

    This method is widely used and similar to the natural process of DNA replication.  It was developed by Frederick Sanger along with Andrew Coulson in 1977. They were awarded the Nobel prize in 1980 on this achievement. Sanger's method now became the standard because of its practicality.
    Sanger's method, which is also referred to as dideoxy sequencing or chain termination, is based on the use of dideoxynucleoside triphosphate (ddNTP's) commonly known as dideoxynucleosides in addition to the normal nucleotides (dNTP's) found in DNA. Dideoxynucleosides are essentially the same as nucleotides except, they contain a hydrogen group on the 3' carbon instead of a hydroxyl group (OH). These modified nucleotides, when integrated into a sequence, prevent the addition of further nucleotides. This occurs because a phosphodiester bond cannot form between the dideoxynucleosides and the next incoming nucleotides, and thus the DNA chain is terminated.

Procedure

  1. Before the DNA can be sequenced, it has to be denatured in single strands using heat because only one strand that acts as a template is required in this procedure. Now the template strand is tagged with a known sequence at 3' end so that a complementary primer can bind on the known sequence.
  2. Once the primer is attached to the DNA, the solution is divided into four tubes each containing a different dideoxynucleoside. Now all these test tubes are placed in the PCR machine so that the sequencing reaction can start. As the DNA is synthesized, nucleotides are added on to the growing chain in place of a normal nucleotide which results in a chain-terminating event. e.g., If we look at only the first tube we might find a collection of three different sized fragments.
  3. Once these reactions are completed, the DNA is once again denatured in preparation for gel electrophoresis. The content of each of the four tubes is run in separate lanes on a polyacrylamide gel in order to separate the different sized bands from one another. After the contents have been run across the gel, the gel is then exposed to either UV light or X-ray, depending on the method used for labeling the DNA.
  4. The sequence read from the gel is complementary to the actual template DNA. Now you can deduce the sequence of template DNA.

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