Question

If you had ddGTP used in sanger sequencing, describe how the nucleotide would be different than dGTP. Why is it important in Sanger sequencing? Be detailed in your repsonse.

Can anyone help with this question?

Answer 1

Before answering these two specific questions, lets discuss about Sanger Sequencing.

It is one of the sequencing methods, which was useful in sequencing large DNA molecules and was developed by Dr. Frederick Sanger.

This method is based on 3 properties of DNA:

1. The chemistry of the nucleotides (monomers of DNA)
2. The mechanism of DNA synthesis from nucleotides by DNA polymerase enzyme.
3. Gel-Electrophoresis; which is used to separate out macromolecules with different size, in this case, DNA sequences of varied sizes.

• The DNA of interest is denatured and the strands are separated.
• The DNA polymerase enzyme require a primer sequence to initiate the process of replication, and also the substrates, which are  deoxy-nucleotides, i.e., dATP, dCTP, dGTP and dTTP.
• Also equal amounts of di-deoxy-nucleotides are added , i.e., ddATP, ddCTP, ddGTP and ddTTP, which lacks the 3'-Hydroxyl groups, compared to their deoxynucleotide counterparts. di-deoxy-nucleotides are also flourescent labelled with a specific color for a particular di-deoxy-nucleotide, which helps in sequencing.
• There are chances that the polymerase encounter either deoxynucleotide or di-deoxy-nucleotide during the chain elongation process. This results in premature termination of the replication process, as di-deoxy-nucleotides lack the nucleophile-3'-Hydroxyl groups which attacks the (phospate moeity) next incoming nucleotide.
• Due to this premature termination of DNA synthesis, DNA strands of different lengths are produced.
• The length of each DNA strand can be compared using electrophoresis, and as the last monomer added is fluorescence-tagged di-deoxy-nucleotide, the sequence composition can also be monitored.

The structures of dGTP and ddGTP are given:

ddGTP will pair with the corresponding dCTP of the parent strand, hence can be used to identify Cytosines in the sequence(s).