Question

What would the results of a Sanger sequencing read look like if you accidentally omitted the deoxyCTP (dCTP) from the reaction?

Multiple Choice

• No Cs could be incorporated into synthesis products, and so only peaks corresponding to the smallest synthesis products - none of which include a C - will appear.

• The results would be normal; dCTP is not required for Sanger sequencing - the only kind of C needed is ddCTP.

• All of the synthesis products would end with ddCTP and so all of the peaks would be the same color.

• No DNA synthesis products ending in ddC would exist, and so those peaks would be absent and the other peaks would simply be closer together to fill the spaces.

• All of the synthesis products would end with C and they would not be fluorescent, so no peaks would appear at all.

• No DNA synthesis products ending in ddC would exist, and so spaces would appear on the read-out where the peaks of C fluorescence would normally be.

Answer 1

• Sequencing refers to the determination of sequence of DNA, RNA or protein.
• DNA sequencing helps in the knowledge of the genetic code, which will further determine the expressed products and their properties.
• Sanger’s method of sequencing is also known as chain termination method.
• In this technique, Dideoxy nucleotides (dd NTPs)- including dd ATP, dd TTP, dd GTP and dd CTP, labelled with colored dye are added, along with d NTPS, DNA polymerase, DNA template and primers.
• When dd NTPS are incorporated, there is no available hydroxyl group, for DNA polymerase, hence chain termination occurs at that point.
• The reaction is carried out in several cycles, and the incorporated dd NTPS, labelled with different dyes, give different peaks in heights.
• If dCTPs are missing, whenever G appers in the sequence, ddCTP will be incorporated instead of dCTP and termination of the sequence will result with all ddCTPs instead of Cs.

Answer:

All of the synthesis products would end with ddCTP and so all of the peaks would be the same color.