Question

The DNA polymerases used over many years for Sanger dideoxynucleotide termination sequencing of DNA have had one common feature: they all were intentionally chosen to lack 3’à 5’ exonuclease activity, the proofreading function that many DNA polymerases carry to maintain high fidelity. Why would having proofreading exonuclease activity within the polymerase be detrimental to the Sanger sequencing technique? If you were to substitute a polymerase with proofreading capability, how might that affect the distribution of the bands of DNA produced in a Sanger sequencing reaction?

Answer 1

During sanger sequencing DNA polymerse used has defective 3'- 5' exonuclease activity.  

In dideoxynucleotides ( ddt) sequencing, dideoxynucleotides ( lack 3' OH) are used in combination with normal deoxynucleotides. During sequencing, synthesis on new DNA strands these dideoxynucleotides incorporated to newly synthsized DNA and lead to chain termination ( because3' OH is required to add coming nucleotides in newly synthesized hain ) . This is actually a misoncopirated nucleotide. In normal DNA replication, this type of misincoporated nucleotides is removed by 3' - 5' exonuclease activity of DNA polymerse. But in the sequencing these terminated synthseized DNA are utilized for detect the DNA stand . So if the DNA polymerse has this exonuclease activity than it will remove these dideoxynucleotides every time and we will not able to deduce sequence of DNA strand. And in the gel we will get whole length band , because every time when ddt incorporated, DNA polymerse remove that and lead to synthesis of normal DNA stand .