VPB 321: Animal Biotechnology (2+1)

DNA Sequencing – Sanger's method (Dideoxy method)

DNA SEQUENCING - DIDEOXY METHOD

Aim: To demonstrate dideoxy method of DNA sequencing

Materials required: dNTPs, ddNTPs, sequencing reagents, materials for PAGE

Method

• The DNA – primer mixture is split into four separate tubes. DNA polymerase and a solution of dNTPs are added to each tube.
• One of the four 2’,3’ – dideoxy – NTPs (ddNTPs) is mixed into each tube, at a concentration 100 times lower than the dNTP concentration.
• DNA polymerase uses the dNTPs in elongate the primer DNA. The occasional incorporation of a ddNTP terminates the polymerization reaction, since the absence of 3’–OH blocks addition of the next nucleotide.
• The ddNTPs are incorporated randomly, resulting in terminated fragments of different lengths.
• The terminated fragments from each of the four reactions are denatured and separated by size using gel electrophoresis.
• The gel is autoradiographed in order to visually detect the labeled DNA fragments. The resulting ladder is read from bottom to top. This represents the complementary sequence of the original DNA template.

Questions

• Define ‘DNA sequencing’
• What are the methods of DNA sequencing?
• What is the principle in using dideoxy nucleotides in Sanger’s method of DNA sequencing?
• Define autoradiography.