Introduction: Protein and DNA are polymeric molecules made up of the monomeric constituent. DNA is made up of 4 different types of nucleotide, A, T, G and C where as protein is made up 20 amino acids. Information present on the DNA is in the form of combination of these 4 nucleotides and responsible for phenotypic changes in an organism. After generation of recombinant DNA, it is also important to confirm its nucleotide sequence before further manipulation in the down-stream processing.
DNA sequencing-Historically there are two methods of DNA sequencing with a similar principle of breaking the DNA (chemical or enzymatic method) into the small fragment followed by separation and analyze them on a high resolution electrophoresis gel.
Di-Deoxy Chain termination or Sanger Methods: This method is originally developed by Frederick Sanger in 1977. In this method, a single stranded DNA is used as a template to synthesize complementary copy with the help of polymerase and in the presence of nucleotides (Figure 37.1). The polymerization reaction contains a primer and nucleotides, 3 normal nucleotides and 2’3’-dideoxynucleotide triphosphate (ddNTPs). When DNA polymerase utilizes ddNTPs as nucleotide, it gets incorporated into the growing chain but chain elongation stops at ddNTPs due to absence of 3’-hydroxyl group. In the typical sequencing reactions, 4 different ddNTPs are taken into the 4 separate reactions and analyzed on high resolution polyacrylamide gel electrophoresis. The ratio of NTPs/ddNTPS is adjusted so that chain termination occurs at each position of the base in the template.
Protocol for Di-deoxy sequencing- There are two protocols people adopt to sequence DNA following di-deoxy chain termination method (Figure 37.2).
Original sanger protocol uses klenow fragment as polymerase for DNA synthesis where as termination protocol uses a T7 polymerase or sequenase. The DNA sequencing by original sanger protocol has following steps:
Step 1: A primer is added and annealed to the 3’ of the DNA template.
Step 2: The radiolabeled 35S ATP to label the primer.
Step 3: The polymerease reaction is divided into 4 reactions.
Step 4: DNA synthesis continues until terminated by the incorporation of the specific ddNTPs (either A, T, G or C).