Question

Sanger method

Answer 1

Principle

The Sanger’s method is a technique for the sequencing of genes. It is also called as dideoxy chain termination method. In this method, nested and labeled set of DNA fragments are generated from a template strand of DNA. The template strand usually is the one which needs to be sequenced. The sequencing is performed by replicating that template strand, and stopping the process at any of the four nucleotides.

Procedure-

1. The experiment begins by the attachment of a DNA primer to the template strand by the process of hybridization.

2. This is followed by the sequential addition of deoxynucleosides triphosphates (dNTPPs) to the primer strand. The reaction is catalyzed by the enzyme DNA polymerase.

3. The reaction mixture also contains dideoxynucleoside triphosphate (ddNTPs) along with usual dNTPs. The ddNTPs cannot form phosphodiester bonds with adjacent nucleotide, and hence replication stops when a ddNTP is incorporated into the strand during replication.

4. Respective ddNTPs of dNTPs are responsible for the termination of the chain at their respective sites. As an example, ddATP would be responsible for the termination of replication of the DNA strand at A site. Similarly ddCTP, ddGTP and ddTTP terminates at C, G and T site respectively.

5. In the process, different strands with difference of one nucleotide are generated. These can be analyzed using electrophoresis and autoradiography.

6. The autoradiogram of the gel would be instrumental in determining the order of bases of complementary strand to that of template strand.