7.2.4 Sequencing DNA: The Sanger Method

Question 1

Sanger method for DNA sequencing

Answer 1

• The sequence of DNA strands can be determined through use of the Sanger method for DNA sequencing, developed by Frederick Sanger.
  • The Sanger method of DNA sequencing involves the use of DNA polymerase to make a strand of DNA that is complementary to the strand of DNA to be sequenced. DNA polymerase builds a nucleotide chain by adding new nucleotides to a DNA strand at its terminal 3’ OH group.

Question 2

dideoxyribonucleotides

Answer 2

• nucleotides that lack two oxygens. Once incorporated into a DNA strand, they can block
  further DNA synthesis because they lack a 3’ OH group. Without a 3’ OH group, the DNA polymerase cannot add another nucleotide.
• A dideoxynucleotide (ddNTP) lacks the 3’ OH group. When it is incorporated into a DNA strand, the strand cannot be lengthened. DNA polymerase will not have the 3’ OH group to add a nucleotide.

Question 3

note

Answer 3

• The diagram to the left shows two nucleotides. Note both nucleotides have an OH group on the 3’carbon.
  DNA polymerase is an enzyme that builds a polymer of DNA by adding a nucleotide to the 3’ OH group of another nucleotide or DNA strand. The dashed line represents the attachment that will be made between the two nucleotides.

Question 4

To determine the sequence of a strand of DNA, four separate test tubes are prepared. Each test tube contains the following:

Answer 4

1. The unknown DNA
2. Regular nucleotides: dATP, dTTP, dGTP, and dCTP
3. DNA polymerase
4. DNA primer (in this case it is ATAA)
   - In each test tube, one of the four different dideoxynucleotides is also added: ddATP, ddGTP, ddCTP, and ddTTP.
   - The complementary strand of DNA is synthesized until a dideoxynucleotide is added. The strands that result from each tube are shown in the lower portion of the diagram.
   - When the reaction mixtures have completed polymerization, the DNA strands from each tube are separated by gel electrophoresis. In this example, note that the shortest fragment was in the GTP lane of the gel. G is the first base in the new DNA strand. The next shortest fragment is in the T lane. Therefore, T is the second base in the DNA strand.
   - Based on the results in the gel, the sequence following the primer is GTACT.

Question 5

The Sanger method relies on

Answer 5

• the ability of gel electrophoresis to separate DNA fragments differing by as little as one nucleotide in length

Question 6

The random incorporation of modified versions of the nucleotides found in DNA is the basis for the Sanger technique. These nucleotides are modified such that:

i. strand synthesis stops when they are incorporated.
ii. they are unable to hydrogen bond with the complementary strand.
iii. they are unable to covalently bond with the next nucleotide in the sequence.

Answer 6

• i, iii

Question 7

Which of the following are necessary ingredients for the Sanger sequencing method?

i. modified nucleotides
ii. unmodified nucleotides
iii. DNA polymerase
iv. single-stranded DNA primer
v. single-stranded DNA to be sequenced

Answer 7

• i, ii, iii, iv, v

Question 8

DNA sequencing techniques

Answer 8

• determine the nucleotide sequence complementary to a single-stranded fragment of DNA beginning at the 3′ end of the DNA molecule being sequenced