Question

Describe the basic principle of traditional Sanger sequencing. You may use drawings to describe your answer.

Answer 1

DNA sequencing is a method of determining the correct sequence of nucleotide in a DNA molecule. Francis Crick suggested the DNA sequencing technique, his theory is that the sequence of nucleotides within a DNA molecule, directly influence the amino acid sequences of proteins.

Two methods of DNA sequencing: a) Traditional method (Sanger sequencing), b) Mordern method (High throughput sequencing).

Traditional method of DNA sequencing is Sanger sequencing. This method is most widely used method for almost 40 years.

The Sanger method depends on a method that binds to a denatured DNA molecule and begin the synthesis of a single-stranded polynucleotide in the presence of DNA polymerase enzyme, using denatured DNA as the template. Sanger sequencing is labour-intensive and time-intensive method.

Sanger sequencing is a method of DNA sequencing which was first commercialised by the Applied Biosystems and is based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication.

Now, Sanger sequencing is mostly used for de novo initial sequencing of a DNA molecule to obtain primary data for an organism.

Ingredients required for DNA sequencing: (i) A DNA polymerase enzyme. (ii) A short piece of single-stranded DNA which binds with the template DNA and acts as a starter for the polymerase, called polymerase.(iii) 4 DNA nucleotides A,T, C,G. (iv) One of the dideoxynucleotide, among ddG, ddA, ddC, ddT.

Through Sanger sequencing, we get high quality sequence for relatively long stretches of DNA i.e, about 900 base pairs. Sanger sequencing is expensive and unfit for large scale project.