VPB 321: Animal Biotechnology (2+1)

SANGER'S DIDEOXY METHOD OF SEQUENCING

• Sanger's dideoxy enzymic method uses specific dideoxy nucleotides (ddNTPs) to terminate enzymically synthesised copies of a template.
• A sequencing primer is annealed to ssDNA template molecule and DNA polymerase extends the primer using ddNTPs.
• This method requires a ssDNA template on which to synthesize complementary copies which means that the DNA has to be cloned into phage vector M13 before sequencing.
• The ssDNA recovered from the phage is annealed to a primer of 15-17 nucleotides which is complementary to the region near the vector- insert junction.
• All sequences cloned into this vector can be sequenced using this universal primer.
• A DNA polymerase enzyme Klenow or T7 DNA polymerase is added to the annealed primer plus template along with a small amount of radioactive labeled ATP.
• It is then divided into 4 tubes each containing a different chain terminator mixed with normal dNTPs (i.e) ( tube C would contain ddCTP and dATP,dCTP,dGTP and dTTP ) in specific rations to ensure only a limited amount of chain termination.
• The 4 sets of reaction products are analyzed by Poly Acrylamide Gel Electrophoresis (PAGE).
• Based on the PAGE analysis, the sequence of the DNA is predicted.
• The PAGE results in the sequences copied from of the template. Hence, to know the original sequence of the DNA, the complementary sequence has to be made.