Sanger Sequencing

 

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Sanger Sequencing   Introduction References

 

Introduction

 

    DNA elongates by adding onto the 3' end of an existing DNA strand. The incoming deoxynucleotide (dNTP) forms an ester linkage between its hydroxyl group, and the hydroxyl group on the existing strand. Incoming nucleotides do not attach in a random order to the existing strand. Their order depends upon the complementary strand’s sequence.

 

    Sanger sequencing takes advantage of dideoxynucleotides (didNTP), which are similar to deoxynucleotides, except they lack a hydroxyl group at the 3’ end of the molecule. They are able to attach to the existing strand, however the strand stops at that point. No other molecules are able to attach onto the dideoxynucleotide.

 

    To sequence a DNA sequence, four reactions are needed – one for each of the four nucleotide bases (Adenine, Guanine, Thymine, and Cytosine). Each reaction contains the template DNA, a primer, DNA polymerase and the four deoxynucleotides with one radioactively labeled. In addition, one type of dideoxynucleotide is added to each.

 

    When the solution is heated to 98 degrees Fahrenheit, the bonds holding the DNA strand together become unstable and release, separating the two strands. After that point, the reaction is cooled down and DNA polymerase begins pairing complementary bases to the denatured strands. The DNA polymerase does not discriminate between dNTP’s and didNTP’s. Randomly, a didNTP is inserted into the sequence, halting all further synthesis. Each of the billions of DNA sequences present are halted at a random point, leaving strands of different lengths as the end product.

 

    Each of the four reactions are then loaded into a different well in a polyacrylamide gel. A current is then run through the gel pulling the bases along towards the positive end. The movement of bases is size dependent with the shortest strands moving the furthest. When viewed under UV light, bands show up where the radioactive bases are. The gel is read from the bottom to the top in order to get the correct sequence of the DNA.

 

 

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References

 

"Sanger Sequencing." Gene Almanac. Dolan DNA Learning Center. 06 Mar. 2008 <http://www.dnalc.org/ddnalc/resources/sangerseq.html>.