DNA sequencing

Question 1

What is sequencing?

Answer 1

The order of nucleotides in a given DNA molecule

Question 2

What is Sanger sequencing?

Answer 2

Dideoxynucleotide chain termination
Developed by Frank Sanger in mid 70’s
ssDNA molecules that differ in length by 1 nucleotide can be separated

Question 3

What are dideoxyribonucleoside triphosphates?

Answer 3

Derivatives of deoxyribonucleoside triphosphates that lack the 3’-OH group

Question 4

Describe how dideoxyribonucleoside triphosphates are used in sequencing

Answer 4

Purified DNA is sequenced in vitro in a mixture that contains ssDNA of the sequence of interest, DNA polymerase, a short primer, and 4 deoxyribonucleoside triphosphates
If a dideoxyribonucleotide analog of one of these nucleotides is also present in the mixture it can become incorporated into the growing DNA chain
As this chain now lacks a 3’-OH, the addition of the next nucleotide is blocked and the DNA chain terminates at that point
To determine the complete sequence of a DNA fragment, the dsDNA is separated from the ssDNA and one of the strands is used as the template for sequencing
4 different chain-terminating dideoxyribonucleoside triphosphates are used in 4 separate DNA synthesis reactions on copies of the same ssDNA template
Each reaction produces a set of DNA copies that terminate at different points in the sequence
The products are separated by electrophoresis in 4 parallel lanes of polyacrylamide gel
The newly synthesised fragments are detected by a label
In each lane, the bands represent fragments that have terminated at a given nucleotide but at different positions in the DNA
By reading off the bands in order, starting at the bottom of the gel and working across all lanes, the DNA sequence of the newly synthesised strand can be determined

Question 5

What are some improvements to Sanger sequencing?

Answer 5

Fluorescently labelled ddNTPs allows all 4 termination reactions to run in one lane